Reprod Dom Anim doi: 10.1111/rda.12333 ISSN 0936–6768

Review Article Does Coxiella burnetii Affect Reproduction in Cattle? A Clinical Update I Garcia-Ispierto, J Tutusaus and F Lo´pez-Gatius Agrotecnio Centre, University of Lleida, Lleida, Spain

Contents Q fever is a zoonosis produced by Coxiella burnetii, a bacterium that is widely distributed worldwide. Domestic ruminants are the most important source of C. burnetii for human infection. In sheep and goats, abortion is the main clinical consequence of infection, yet the symptoms described in cattle have so far been inconsistent. Q fever has been also scarcely reported in cattle, most likely because of its difficult diagnosis at the farm level and because of the many existing responsible C. burnetii strains. In this report, the effects of C. burnetii infection or Q fever disease on the reproductive behaviour of dairy cattle are reviewed, with special emphasis placed on the scarcity of data available and possible control actions discussed.

Introduction Q fever is a disease produced by the widespread bacterium, Coxiella burnetii, known to affect humans (Marrie 1990), farm or wild animals (Babudieri 1959) and arthropods (Maurin and Raoult 1999). Over the period 2007–2010, an outbreak of the disease was produced in the Netherlands (Georgiev et al. 2013), and 4026 human cases of infection were reported. In most species, infection is usually asymptomatic. In humans, it usually occurs as a flu-like syndrome, although some patients may also present with endocarditis, hepatitis, osteoarticular infection or spontaneous abortion (Parker et al. 2006). For the European Union to record a case of Q fever in humans, one of the following criteria needs to be fulfilled: clinical symptoms of fever, pneumonia or hepatitis, a laboratory diagnosis (isolation of C. burnetii, detection of C. burnetii DNA or a specific antibody response) or epidemiological findings (exposure to a common source or animal-tohuman transmission) (EFSA 2010a,b). The consequences of Q fever infection in humans are therefore well established and described. Yet, in domestic ruminants, which act as the main reservoir of C. burnetii for human infection, the situation is far less well understood, and although it is well established that abortion is the main sign of infection in sheep and goats (Masala et al. 2004; Arricau-Bouvery and Rodolakis 2005; S anchez et al. 2006; Wouda and Dercksen 2007), the effects of bovine coxiellosis described in the literature have been conflicting. In the cow, although its prevalence could be considered high (20% and 38% at the individual and herd level, respectively) (Guatteo et al. 2011), Q fever is often described as asymptomatic (Ortega-Mora 2012). Factors linked to the disease in cattle have been infertility, abortion (To et al. 1998; Bildfell et al. 2000) and metritis and mastitis (Arricau-Bouvery and Rodolakis 2005; © 2014 Blackwell Verlag GmbH

Barlow et al. 2008).Yet, correlation between Coxiella seropositivity and fertility and a low abortion risk has also been reported (Lo´pez-Gatius et al. 2012; GarciaIspierto et al. 2013). This discrepancy is likely due to the difficulty in diagnosing Q fever (not C. burnetii infection) at the farm level. Furthermore, the type of C. burnetii strain involved could add to this confusion. The aim of this review was to highlight the important implications of C. burnetii infection or Q fever disease in dairy cattle reproduction and to discuss possible control measures.

Clinical Relevance of Coxiella burnetii Infection in Dairy Herds The foetal and newborn periods Q fever has been related to stillbirth and aborted foetuses (Arricau-Bouvery and Rodolakis 2005). In several studies, C. burnetii has been PCR detected in the placenta as a placentitis confirmed by histology. In addition, a large number of publications have described the presence of the bacterium in placental and foetal tissues by immunohistochemistry (van Moll et al. 1993; Bildfell et al. 2000; Hansen et al. 2011). Recently, Muskens et al. (2012) reported the presence of the bacterium by PCR in 9 of 100 aborted foetuses or stillborn calves. A question that is still under debate is whether a rise in bulk tank milk (BTM) antibodies may be linked to an increased risk of abortion. Nielsen et al. (2011) detected no clear relationship between BTM antibodies against C. burnetii and perinatal mortality, but the risk of perinatal death was twofold higher in herds showing high levels of BTM antibodies compared with the remaining herds. Infection mechanisms are still unknown, although transplacental infection of foetuses seems possible (Angelakis and Raoult 2010). Post-partum diseases Abortion, stillbirth and placenta retention have been related to subsequent post-partum diseases such as metritis or endometritis in lactating dairy cows (LeBlanc 2008). Yet, in C. burnetii infection, there is no experimental evidence pointing to a direct link between C. burnetii infection and post-partum disease (Agerholm 2013). Placenta retention Positive correlation has been reported between dam serology and retained foetal membranes (Vidic et al.

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1990). In a recent study (Lo´pez-Gatius et al. 2012), previous retained membranes were correlated with C. burnetii seropositivity. Yet, this study was unable to identify a link between placenta retention and subsequent decreased fertility as has been extensively reported. Thus, the implications of this finding are still to be clarified. Mastitis It is widely assumed that C. burnetii is shed via the milk (Willems et al. 1994; Lorenz et al. 1998), but few studies have related the presence of the bacterium with mastitis. Some reviews include C. burnetii among the miscellaneous known organisms that may cause mastitis (Philpot and Pankey 1975), while another broad review of bacterial aetiologic agents of mastitis fails to mention it (Watts 1988). In a more recent report, it was suggested that the prevalence of C. burnetii infection was higher among dairy cattle with reproductive problems including mastitis (To et al. 1998). Furthermore, the PCR finding of C. burnetii in milk associated with increased postpartum somatic cell counts (Barlow et al. 2008) as a tool for mastitis control suggests that this bacterium leads to unidentified economic losses in dairy farms. Further research is needed to evaluate the consequences of C. burnetii infection on the udder health of the dairy cow. Metritis Metritis has been defined as an abnormally enlarged uterus, a fetid watery red brownish uterine discharge associated with signs of systemic illness and fever within 21 days in milk (DIM) (Sheldon et al. 2006). This postpartum condition is among the most frequent illness to affect the subsequent reproductive performance of dairy cows. Thus, determination of the effect of C. burnetii infection seems crucial. Some authors have reported an increased prevalence of metritis in seropositive animals (To et al. 1998), while others have detected no such relationship (Muskens et al. 2011). The detection of C. burnetii in the uterus is not surprising as it may also be present in the genital tract of healthy animals (Luoto and Huebner 1950; Guatteo et al. 2006, 2007; Garcia-Ispierto et al. 2013; Tutusaus et al. 2013). Thus, the link between these two factors may have little significance. Further appropriately designed studies and proper definitions are needed for an infection as widespread as Q fever. Endometritis Endometritis, or inflammation of the endometrium without affecting the remaining uterus layers (LeBlanc et al. 2002; Sheldon et al. 2006), is often misdiagnosed when the cervix closes rapidly after parturition (Kasimanickam et al. 2004), especially since the definition and diagnosis of subclinical endometritis is still under discussion (Lewis 1997; Barlund et al., 2008; Senosy et al., 2009; Lopez-Helguera et al., 2012). It is therefore difficult to relate C. burnetii infection to this uterine disorder. While some authors have observed differences between seropositive or C. burnetii shedding animals

I Garcia-Ispierto, J Tutusaus and F Lo´pez-Gatius

(Woernle and Mu¨ller 1986), other authors have not been able to do so (Sting et al. 2000). In a recent study, Garcia-Ispierto et al. (2013) showed that seropositive cows exhibited a lower risk of suffering endometritis diagnosed by ultrasonography than seronegative animals. To establish a clear relationship between this infection and endometritis, scientists first need to reach a consensus about what subclinical endometritis entails and define a C. burnetii-infected animal. In effect, differences among the studies could be the outcome of the various existing definitions of endometritis, or due to seropositive animals being protected against coxiellosis if they have recently suffered an acute infection. Conception rate Since the 1980s, a decline in the fertility of dairy cattle has been observed in parallel with an ever-increasing milk production (Lo´pez Gatius, 2003; Lo´pez-Gatius et al. 2006). The reason for this decline is multifactorial and involves genetic, nutrition, production and management factors. The effects of infectious disease on the conception rate need to be carefully assessed. Several post-partum factors such as placenta retention, metritis and endometritis have already been reported to reduce conception rates (Gro¨n et al. 1990; Fourichon et al. 2000; Lo´pez-Gatius et al. 2006; Mee, 2008; Bell and Roberts 2007). Coxiella burnetii infection has been often linked to infertility (Krauss et al. 1987; Aitken 1989; To et al. 1998). Yet, in a recent study analysing a study population of 781 parous cows showing 50% seroprevalence against C. burnetii, we could demonstrate that early fertile cows (cows becoming pregnant before Day 90 of lactation) were more likely to be seropositive than the remaining animals (Lo´pez-Gatius et al. 2012). Again, these statements should be carefully interpreted. Several factors can modify the results of investigations designed to correlate C. burnetii infection with the conception rate: (i) the definition of infection, that is, should we define a Coxiella-infected animal according to its seropositivity or shedding status?, (ii) the circulation of different C. burnetii genotypes, that is, what effect has the C. burnetii strain on the cow? and (iii) the monitorization or not of other factors traditionally affecting the conception rate such as previous postpartum diseases, days in milk, milk production, the inseminating semen, the inseminator or the season, among others. We may assume that the real clinical impacts of C. burnetii infection on conception rate in dairy herds have not been adequately addressed in the different studies. In addition, the high costs of tests (for example, PCR for all routes of shedding such as milk, faeces and vaginal fluid during the post-partum and insemination periods) question the real need for these kinds of study. Finally, large study populations are needed to examine the consequences of a given factor on conception rate. Pregnancy loss Pregnancy loss is the effect attributed to Q fever in sheep and goats, especially during the third trimester of © 2014 Blackwell Verlag GmbH

C. burnetii Clinical Effects on Reproduction in Dairy Cattle

gestation (Masala et al. 2004; Woldehiwet 2004; Arricau-Bouvery and Rodolakis 2005; Sa´nchez et al. 2006; Wouda and Dercksen 2007; Jones et al. 2010). In cattle, results have again been inconclusive. Thus, one study was able to relate C. burnetii in foetuses or the seropositivity of dams to pregnancy loss, yet experimentally the bacterium could not be associated with abortion (Agerholm 2013). When dams were inoculated with C. burnetii Nine Mile strain, stillbirth or pregnancy loss could not be consistently related to C. burnetii (Plommet et al. 1973; Behymer et al. 1976). More recently, several studies have detected no relationship between C. burnetii infection of the dam and pregnancy losses (Lange et al. 1992; Tramuta et al. 2011; Lo´pezGatius et al. 2012; Yang et al. 2012). As biosecurity measures do not allow the inoculation of C. burnetii in commercial dairy herds, the only possible investigation strategy consists of epidemiological studies. As has been noted for the pregnancy rate, the main way of determining whether C. burnetii infection is a risk factor for abortion is to monitor all other factors possibly related to pregnancy loss, such as management, environmental and cow factors, as well as concurrent infectious diseases. These factors have not been controlled in any relevant study published to date.

Aspects of Laboratory Diagnoses Serology Serology is the cheapest laboratory method currently available to diagnose C. burnetii infection in a herd. Seroprevalence in dairy cows ranges from 37% to 100% in herds in Europe (EFSA 2010a,b). ELISA is the most commonly used assay for screening herds, but its interpretation at the individual level is difficult. To date, we are unaware of the exact time of seroconversion or how long antibodies persist in the cow (Kennerman et al., 2010). Furthermore, although there is positive correlation between serology and shedding (Guatteo et al. 2007; Courcoul et al. 2010), the presence of seronegative animals that shed C. burnetii and seropositive animals that do not (Guatteo et al. 2007; Rousset et al. 2009; Hansen et al. 2011) questions the significance of serology in dairy cows. Despite this, studies on the dynamics of antibody titres during the productive life of the cow will help interpret serological data, and numerous questions regarding serology interpretation and its relationship with clinical symptoms remain unsolved. Calves The literature lacks extensive information on C. burnetii antibody levels in the neonate. Vertical transmission of C. burnetii has not been demonstrated. In a recent study, we noted that the pre-colostral antibody response was not detectable in calves born from dams with C. burnetii-qPCR-positive cotyledons (Tutusaus et al. 2013). After the intake of the colostrum of seropositive mothers, all calves seroconverted. What now needs to be addressed is whether these seronegative calves born from infected dams show the presence of the bacterium

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by PCR. If not, we will need to determine the mechanism that does not allow bacteria to cross the placenta to the foetus in the case of live newborns. It could be that failure of this mechanism determines abortion or stillbirth. Studies performed in mice (Baumga¨rtner and Bachmann 1992) suggest that foetus–placental union resists C. burnetii vertical infection and that the infection of the newborn is established by aerosol inhalation at the moment of parturition. In contrast, if vertical transmission is possible, this could be an indication of persistent infection due to immune tolerance to an early in utero infection, as proposed for other infections such as bovine viral diarrhoea virus (Houe et al. 1995) or Mycobacterium avium subsp. paratuberculosis (Whittington and Windsor 2009). Probably, both serology and molecular techniques are needed to better understand the effects of C. burnetii infection on the newborn calf. Heifers Little is known about seroprevalence and seroconversion in heifers. Czaplicki et al. (2012) demonstrated that the risk of serconversion was higher in heifers drinking water from a watercourse and lower in heifers tie and free stalling. In another study performed using phasespecific ELISA (PhII produced early after infection and PhI delayed after PhII), it was found that PhI /PhII+ always became negative in these animals (Bo¨ttcher et al. 2011). This finding could be explained by maternally derived antibodies, which tend to disappear in heifers. What remain unclear is when maternal antibodies decrease and when those heifers are infected. Primiparous and multiparous cows Pregnant cows can show a very stable pattern of C. burnetii antibodies throughout gestation along with a post-partum decrease in serum antibodies (GarciaIspierto et al., 2011s), probably due to their shunting to colostrum prior to calving. This post-partum antibody decline was more clearly observed in primiparous cows, which showed higher antibody levels compared with multiparous cows throughout gestation (Garcia-Ispierto et al., 2011). In effect, although there is a higher probability of having come into contact with the bacterium in multiparous cows, in a recent study, multiparous cows showed a lower risk of being seropositive than primiparous cows (Garcia-Ispierto et al., 2011). This apparent contradiction may not be difficult to interpret in the case of dairy cows. In dairy herds, cows with reproductive disorders are culled. Thus, it could be that C. burnetii-seropositive cows were culled in higher proportions than their seronegative partners. No existing report has examined the seroprevalences of culled cows. Seroconversion or seronegativization is not a common event in cattle (Guatteo et al. 2007; Garcia-Ispierto et al., 2011). In primiparous cows, seroconversion mainly occurs during their first 90 days in milk (Nogareda et al. 2012), probably revealing a C. burnetii transmission pattern in the younger animals. The

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presence of a small number of multiparous cows that fail to seroconvert and remain seronegative over time has also been reported (Nogareda et al. 2012). The reason for some cows not developing a humoral response is still unknown. Shedding patterns Shedding of bacteria occurs through milk, faeces and vaginal mucous. A recent study showed that dairy cow shedders with antibodies shed for a longer period of time than shedders without antibodies (Courcoul et al. 2010). Despite this, serology is not a completely reliable screening test for the detection of shedders within a herd (Natale et al. 2012), and the PCR investigation of all possible transmission routes is necessary to understand C. burnetii infection. Furthermore, animals do not shed the bacterium continuously, and intermittent shedding is the common status of the infected cow (Guatteo et al. 2007). At the clinical level, this is practically and economically unviable. Shedding of C. burnetii via any of the three routes during parturition and the third trimester of gestation is maximized due to recrudescence of the bacterium (Harris et al. 2000). The immunosuppression status of the cow during parturition due to high plasma concentrations of progesterone and cortisol may explain this increase in the number of cows shedding in these periods (Lewis 2004). During the post-partum, shedding animals fall in numbers until 90 days in milk, probably due to recovery of their immune status (Harris et al. 2000; Lewis 2004). A persistent shedding pattern has been determined in milk (Guatteo et al. 2007), but not in vaginal mucous (Guatteo et al. 2007) or faeces (Guatteo et al. 2007). These findings indicate that the digestive tract, uterus and vaginal environment may be less attractive for the bacterium than the mammary gland. The clinical repercussions of this have yet to be determined. In humans, the shedding of C. burnetii starts before seroconversion (Parker et al. 2006). In dairy cattle, this has not yet been demonstrated so efforts are needed to determine the exact relationship between shedding patterns and serology. Endocrinology patterns based on serological findings Little is known about the repercussions of C. burnetti seropositivity on hormonal patterns during pregnancy in cows. To the best of our knowledge, only one study has examined this issue. Garcia-Ispierto et al. (2010) determined that the presence of C. burnetii antibodies leads to a decrease in pregnancy-associated glycoproteins (PAG) during the second trimester and increased cortisol concentrations at 180 days of pregnancy. These findings may reflect effective placental damage. Furthermore, when there was concomitant infection with Nespora caninum, progesterone levels during the whole of gestation were elevated (Garcia-Ispierto et al. 2010). Perhaps, the key to knowing the reproductive consequences of a subclinical infection such as Q fever is to determine its effects on endocrinology patterns both in pregnant and non-pregnant animals.

I Garcia-Ispierto, J Tutusaus and F Lo´pez-Gatius

Possible Role of Coxiella burnetii Strain in Reproductive Performance Strain is important in C. burnetii transmission to humans. More than 50 years ago, six genomic groups of C. burnetii were defined: groups I to III contain strains isolated from ticks, human acute Q fever cases and livestock abortions (Russell-Lodrigue et al., 2009); strains in groups IV and V have been related to livestock abortions and chronic endocarditis or hepatitis (RussellLodrigue et al., 2009); and group VI are infectious yet avirulent strains in rodents (Russell-Lodrigue et al., 2009). In 2007, interest in Q fever as a potential public health threat increased following the Netherlands outbreak. This outbreak was retrospectively associated with an increased dairy goat farm density in the area. Despite this, farming or other occupational activities could not be documented for all human Q fever cases reported during the outbreak (Maurin and Raoult 1999). Thus, virulence factors associated with circulating isolates have been questioned (Russell-Lodrigue et al., 2009). Genotypic characterization of human and animal C. burnetii isolates demonstrated the polyclonal nature of the Dutch outbreak. Recently, Jado et al. (2012) demonstrated that C. burnetii genotypes are highly variable in Spain. The only cattle genotype (related to group VI Dugway) was not found in human clinical samples, while sheep, goats, wild boar, rats and ticks shared genotypes with the human population. Whether this also occurs in other countries or, on the contrary, cattle can transmit the bacterium to humans needs to be investigated. Identifying the effects of different genotypes of bacteria could help determine why some studies have detected compromised reproductive performance in C. burnetiiinfected herds, while others have not.

Does Vaccination Against Coxiella burnetii Improve Reproductive Performance? Today, measures to control C. burnetii infection on a farm consist of two main strategies: (i) antibiotherapy or (ii) vaccination. The former, although demonstrated to reduce shedding in cattle, does not reduce abortion or prevent shedding (Durand 1993; Rodolakis 2009; Angelakis and Raoult 2010; Taurel et al. 2012b) and is not economically viable in dairy herds. In effect, milk obtained following antibiotherapy has to be removed from the food chain (European Commission 1996). Thus, vaccination against C. burnetii is the only possible solution to prevent bacterial shedding in a herd. Two vaccine types against C. burnetii have been developed: phase I and phase II. The phase I vaccine seems the most protective, inducing seroconversion and reducing bacterial shedding and abortion rates in seronegative and/or PCR-negative goats (de Cremoux et al. 2012). In dairy cattle, a Th2 immune response and reduced shedding has been observed only in non-pregnant animals that are seronegative and/or PCR negative (Guatteo et al. 2008). When used in infected animals during the peri-insemination period, vaccination does not prevent C. burnetii shedding (Guatteo et al. 2008; Rousset et al. 2009) questioning vaccination in adults. Yet, © 2014 Blackwell Verlag GmbH

C. burnetii Clinical Effects on Reproduction in Dairy Cattle

nulliparous heifers, most of which are considered to be non-infected animals (Taurel et al., 2011, 2012a), are a common target population for vaccination. Because of farm management policy, vaccination postAI is sometimes difficult and requires additional management efforts. Recently, two studies have examined the use of this vaccine during the dry period. Although it did not reduce shedding during post-partum period (Tutusaus et al. 2014), the vaccine proved safe in that it did not increase the abortion rate and was able to improve the subsequent fertility of the herd, especially when applied to C. burnetii-seronegative animals (Lo´pez-Helguera et al. 2013). The question that arises is how vaccination against C. burnetii increases reproductive performance. Two hypotheses emerge: (i) vaccination protects seronegative animals against infection with C. burnetii or (ii) non-specific immunostimulation after vaccination is beneficial for the animal. It is therefore probably better to vaccinate heifers (usually seronegative animals) and pregnant cows to increase subsequent reproductive performance in an already infected herd.

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performance. Specific genotypes of the bacterium could explain the current controversy over this issue (Jado et al. 2012). To monitor C. burnetii infection, both serology and shedding patterns should be analysed in individual cows in a herd. But the elevated costs of these tests make it impractical to monitor the disease. Despite this, more serology/prevalence studies, including the determination of different C. burnetii genotypes are needed worldwide to establish how Q fever can affect the reproductive performance of cattle. Vaccination of heifers seems the only possible way to control Q fever in dairy herds. Conflict of interest None.

Author contributions Garcia-Ispierto wrote the manuscript; Tutusaus helped with data collection; Lo´pez-Gatius revised the manuscript.

Conclusions The presence of C. burnetii in dairy herds has been not yet clearly demonstrated to negatively affect reproductive

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Submitted: 14 Jan 2014; Accepted: 15 Apr 2014 Author’s address (for correspondence): I Garcia-Ispierto, Agrotecnio Centre, University of Lleida, 25198 Lleida, Spain. E-mail: [email protected]