Accepted Manuscript

Q fever and prevalence of Coxiella burnetii in milk

Andreana Pexara, Nikolaos Solomakos, Alexander Govaris

PII: S0924-2244(17)30608-8
DOI: 10.1016/j.tifs.2017.11.004
Reference: TIFS 2114

To appear in: Trends in Food Science & Technology

Received Date: 16 September 2017

Revised Date: 6 November 2017
Accepted Date: 9 November 2017

Please cite this article as: Pexara, A., Solomakos, N., Govaris, A., Q fever and prevalence of Coxiella
burnetii in milk, Trends in Food Science & Technology (2017), doi: 10.1016/j.tifs.2017.11.004.

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 ACCEPTED MANUSCRIPT
1 Q FEVER AND PREVALENCE OF COXIELLA BURNETII IN MILK

2 Pexara Andreana*, Solomakos Nikolaos, Govaris Alexander

3 Laboratory of Hygiene of Foods of Animal Origin, Faculty of Veterinary Medicine,

4 University of Thessaly, 224 Trikalon Str., 43100 Karditsa, Greece.

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5

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6 *Corresponding author

7 Laboratory of Hygiene of Foods of Animal Origin

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8 Faculty of Veterinary Medicine

9 University of Thessaly

10
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Address: 224 Trikalon Str., 43100 Karditsa, Greece
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11 Telephone: 0030 24410 66023, Fax: 0030 24410 66087

12 E-mail: apexara@vet.uth.gr
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13
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14 ABSTRACT
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15 Background

16 Q fever is a zoonosis caused by Coxiella burnetii. In humans, although it has been

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17 predominantly considered an occupational hazard, in the last decades, Q fever outbreaks

18 have also been reported in various countries, indicating its importance as an emerging
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19 public health threat. Domestic ruminants are considered as the most important sources
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20 of human infection. In fact, both symptomatic and asymptomatic infected ruminants

21 shed the bacterium into the environment with birth products, but also in urine, faeces,

22 vaginal mucus and milk. Q fever in humans is mainly asymptomatic, but it also may

23 manifest itself as an acute or chronic disease with long-term sequelae. Inhalation of

24 infectious aerosols usually causes the disease in humans, but the presence of C. burnetii in

25 raw milk raises concern over the role of milk as a source of infection.
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26 Scope and Approach

27 In this review data on Q fever in humans are summarized and the possible transmission of

28 C. burnetii to humans by consumption of unpasteurized milk is discussed. In addition, an

29 overview of the published data on the prevalence studies of C. burnetii in raw milk in

30 various countries is provided.

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31 Key Findings and Conclusions

32 Recent surveys conducted in many countries have revealed that the prevalence of C.

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33 burnetii in raw milk can vary over a wide range from 0% to as high as 95%. Based on recent

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34 survey data, the risk of C. burnetii infection by consuming unpasteurized milk and raw

35 milk products cannot be considered negligible.

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36
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37 Keywords: Coxiella burnetii; Q fever; Milk; Zoonoses; Public health

38
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39 Introduction
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40 Q fever is a zoonosis with almost worldwide distribution caused by Coxiella burnetii,

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41 an obligatory intracellular Gram-negative bacterium belonging to the family of

42 Coxiellaceae (Angelakis & Raoult, 2010). C. burnetii is usually grown in two stages with two
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43 different morphological types namely the large-cell variant (LCV) and the small-cell

44 variant (SCV). The LCV is obviously the larger and is the metabolically active intracellular
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45 form of C. burnetii. The SCV represents the small morphological variant of the bacteria
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46 which are released during lysis of infected cells, resulting in the spore-like form found in

47 the environment. Due to the resistant SCV morphotype, C. burnetii is stable in the

48 environment and resistant to physicochemical stresses such as disinfectants,

49 dehydration, irradiation or osmosis. Thus, the bacterium can survive for a long time in

50 dairy and meat products as well as aborted fetuses, manure, wool, animal feed,

51 equipment and clothes (EFSA, 2010). For example, C. burnetii may live 1 year in wool, 4
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52 months in dust, and 1 month in dehydrated sputum (NABC, 2010). The SCV is the

53 infectious form phagocytosed by macrophages during early infection and the pathogen

54 form associated to food-borne risk (EFSA, 2010). Monocytes and macrophages are the

55 major targets of C. burnetii and spread in the host tissues. After an acute infection, C.

56 burnetii can survive for a long time or even permanently in the macrophages (Gwida, El-

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57 Ashker, & Khan, 2012).

58 C. burnetii is considered a ubiquitous zoonotic contaminant since it can cause

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59 infections in several animal species, wild and domestic animals, birds, reptiles, and ticks.

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60 Cattle, sheep and goats are the most common reservoirs for this pathogen and main

61 sources of human infection (Angelakis & Raoult, 2010). In animals, the infection is usually

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62 asymptomatic, but clinical manifestations of the disease are associated with reproductive
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63 disorders may occur. Both symptomatic and asymptomatic infected ruminants shed C.

64 burnetii in large amount in the environment. C. burnetii is mainly shed during and after
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65 parturition or abortion in birth products (placenta, birth fluids) but also in urine, faeces,
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66 vaginal secretions and milk of infected animals (van den Brom et al., 2012).
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67 The clinical appearances of Q fever in people are variable. In fact, it can be

68 asymptomatic or it can manifest itself in acute or chronic form. Acute Q fever in people
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69 generally shows as an asymptomatic or mellow flu-like infection (Maurin & Raoult, 1999).

70 In most cases, a little minority of patients show a genuine illness which can prompt
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71 genuine intricacies. In some people, the disease can lead to a chronic infection with long-
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72 term sequelae (Arricau-Bouvery & Rodolakis, 2005).

73 Q fever was considered predominately an occupational hazard and direct contact

74 with ruminants may be strongly associated with the disease in humans (Angelakis &

75 Raoult, 2010). A few years ago, Q fever attracted international attention due to the

76 largest-ever recorded outbreak in the Netherlands involving over 4.000 reported human

77 cases during 2007-2010 (Vanderburg et al., 2014). Also, over the past 20 years, Q fever
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78 outbreaks in human have been reported in various countries (Bulgaria, Bosnia and

79 Herzegovina, Germany, USA) (Kamenov & Tiholova, 2004; Sukrija et al., 2006; Martinov,

80 2007; Gilsdorf et al., 2008; Bjork et al., 2013). Although in large outbreaks, sheep and

81 goats have been considered as the source of infection (Arricau-Bouvery & Rodolakis,

82 2005), humans’ outbreaks occurred both in rural and urban areas and many cases of Q

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83 fever were unrelated to occupation (EFSA, 2010). These outbreaks highlighted the

84 importance of Q fever as an emerging public health threat.

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85 Moreover, the high distribution of C. burnetii in productive animals necessitates the

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86 evaluation of the pathogen presence in foods of animal origin and the associated

87 potential risk to public health (Pexara, Solomakos, & Govaris, in press). Among the foods

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88 of animal origin, the raw milk is the most significant source of C. burnetii (EFSA, 2010;
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89 Gale, Kelly, Mearns, Duggan, & Snary, 2015). The presence of C. burnetii in milk raises

90 concern over the role of raw milk or raw-milk products as possible infection routes of this
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91 zoonotic pathogen into humans.

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92 This article provides data on Q fever in humans and addresses the possible
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93 transmission of C. burnetii to humans by consumption of unpasteurized milk. An overview

94 of the published data on the prevalence studies of C. burnetii in the raw milk is also
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95 provided in various countries.

96
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97 Q fever in humans
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98 Infection Sources and Transmission

99 Humans are often very susceptible to Q fever and even 10 cells or fewer of C. burnetii

100 may cause infection (CDC, 2015). Since C. burnetii may be found in several mammalian

101 species, and non-mammalian species such as birds, reptiles and ticks, the source of C.

102 burnetii infection in humans is often not clear (Stein & Raoult, 1999). The primary

103 reservoirs of the pathogen are sheep, goats and cattle (Maurin & Raoult, 1999). Although
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104 in these animals C. burnetii infection is common, the clinical disease is rare. In small

105 ruminants, Q fever is mostly connected with sporadic abortions or episodes of premature

106 births and dead. In cattle, the described symptoms have so far been inconsistent. Many

107 studies have reported that Q fever plays a role in infertility or problems such as metritis,

108 mastitis and a low abortion risk (Lopez-Gatius, Almeria, & Garcia-Ispierto, 2012). However,

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109 the presence of C. burnetii in dairy herds has been not yet clearly demonstrated to cause

110 a negative reproductive performance. Contrary, Garcia-Ispierto et al. (2013) reported that

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111 seropositive shedding cows showed a better reproduction performance than healthy

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112 cows.

113 It is very important to note that the environment is polluted with C. burnetii since

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114 both symptomatic and asymptomatic animals shed the pathogen. C. burnetii is usually
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115 found in the uterus and mammary glands of infected ruminants and can remain for more

116 than 20 months (EFSA, 2010). High amounts of C. burnetii cells are usually shed during
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117 abortion or parturition of infected ruminants, while placentas of infected small ruminants
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118 may have over 109 doses of hamster infection (Arricau-Bouvery & Rodolakis, 2005).
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119 The pathogen can be also excreted in milk, faeces and urine of animals. C. burnetii

120 shedding in milk can persist for several months and is longer in goats than in sheep
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121 (Arricau-Bouvery & Rodolakis, 2005). Goats and cows mostly shed C. burnetii in milk and

122 vaginal mucus whereas sheep shed the pathogen cells at a higher number in faeces
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123 (Rodolakis et al., 2007). C. burnetii has been also isolated in bull semen (Kruszewska &
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124 Tylewska-Wierzbanowska, 1997).

125 Aborting ruminants have been considered as the most significant sources of Q fever

126 in human cases, while infected sheep and goats were several times accused of large C.

127 burnetii outbreaks (Arricau-Bouvery & Rodolakis, 2005). In several also cases of human

128 outbreaks other species such as pigeons, rabbits, dogs or cats were also identified as the
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129 causative agents of infection (Stein & Raoult, 1999). Dogs are usually contaminated by

130 eating the infected placentas (Maurin & Raoult, 1999).

131 Ticks appear to play an important role in enzootic transmission cycles in wild

132 vertebrates (rodents and lagomorphs), while wild birds can also spread infections to

133 domesticated ruminants. However, the role of ticks in the transmission of the disease to

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134 humans is debated and varies according to certain countries cases (EFSA, 2010)

135 Humans can be infected by direct contact with infected animals particularly during

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136 abortion and parturition, milk, urine, faeces, or semen from an infected animal (Arricau-

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137 Bouvery & Rodolakis, 2005). In humans, the airborne pathway is the main route of

138 transmission of the Q fever. The inhalation of birth fluids or placental materials has also

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139 identified as sources of transmission to humans (CDC, 2015). The bacterium can become
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140 airborne, traveling on wind currents for miles, and cause outbreaks (EFSA, 2010). The

141 Centers for Disease Control classified C. burnetii as a category B biologic warfare and
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142 bioterrorism agent due to a low dosage of infection, coupled with the organism's ability
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143 to cause debilitating disease and high levels of resistance to various means of inactivation
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144 (CDC, 2015).

145 Uncommon human-to-human transmissions from an infected woman placenta, blood

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146 transmission and bone marrow transplantation from asymptomatic patients were also

147 reported (Hogema et al., 2012). Sexual transmission is also possible (Domingo et al.,
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148 1999). Rare cases of autopsies and obstetrical operations in nosococomial infections
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149 were previously reported (Raoult & Stein, 1994). C. burnetii has been isolated from

150 human breast milk but no cases have been documented via lactogenic transmission

151 (Kumar, Yadav, & Kakkar, 1981).

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153 Epidemiology
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154 Farmers, veterinarians, abattoir workers, animal handlers are usually prone to a Q

155 fever infection. Nevertheless, community outbreaks in persons with no contact to

156 animals have also been described (Vellema & van den Brom, 2014).

157 Pape et al. (2009) reported that individuals aged between 30 and 50 years old

158 showed an increased risk to C. burnetii infection. According to EFSA data in 2009 the

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159 highest notification rate of human Q fever in Europe was among the group of 45 to 64

160 years old followed by the groups of 25 to 44 years old and over 65 years old (EFSA, 2011).

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161 This age-related increase of Q fever incidence may be attributed to a higher contact with

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162 farm animals during countryside travel or rural activities (Pape et al., 2009). Although Q

163 fever cases in children have been reported, the disease among children under the age of

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164 15 years old is rare (Pape et al., 2009; EFSA, 2011). The infection rate seems to be higher in
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165 men than in women, and a rate ratio of almost 2:1 (Pape et al., 2009; EFSA, 2010). This

166 could be explained by the fact that men involvement in farming activities is higher than
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167 that of women (Maurin & Raoult, 1999). Leone et al. (2004) reported that women may be
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168 also protected by female hormones.

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169 Information on seasonality of infection reveals that the increased incidence is linked

170 to lambing season. In many European countries, the highest numbers of cases were
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171 reported during summer due to spring lambing season (EFSA, 2010). In general, the

172 distribution by the date of notification of Q fever cases is not very useful for the date
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173 consideration of the illness onset (EFSA, 2011).

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174

175 Clinical manifestations

176 The main characteristic of Q fever in human is its clinical polymorphism. Following

177 primary infection, more than half of the patients remain asymptomatic (Karakousis &

178 Trucksis, 2006). Q fever may manifest as acute Q fever or chronic Q fever with long-term

179 sequelae (Raoult et al., 2000).

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180 Acute Q fever usually develops as a non-specific self-limiting febrile illness,

181 pneumonia, or hepatitis (Karakousis & Trucksis, 2006). It has been reported that only 2%

182 to 4% of subjects with symptomatic acute Q fever is admitted to hospital. In fact, Q fever

183 is usually not diagnosed due to the unspecific symptoms (Tissot-Dupont, Vaillant, Rey, &

184 Raoult, 2007).

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185 The incubation period of acute Q fever may be 7 – 40 days or longer and depends on

186 the inoculation dose of C. burnetii (Maurin & Raoult, 1999; Vellema & van den Brom,

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187 2014). In the first described cases, fever and headache were the most prominent

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188 symptoms. Typically, a high fever of 39-40°C reaches a plateau in 2-4 days and then, after

189 5-14 days, the temperature returns to normal (Angelakis & Raoult, 2010). Q fever is

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190 classically observed as atypical pneumonia. However, a very common form of infection by
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191 C. burnetii is also hepatitis; it may be expressed as infectious-like hepatitis or fever of

192 unknown origin (FUO) with characteristic hepatic granulomas on liver biopsy.
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193 Myocarditis, pericarditis, meningoencephalitis and skin rash are rarely observed
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194 (Angelakis & Raoult, 2010).

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195 Chronic Q fever develops after acute Q fever in about 1–5% of the cases and mostly

196 manifests itself within the first year after infection, but the disease can also present after
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197 several years after the initial infection. Mortality occurs in 1 to 11% of patients with chronic

198 Q fever (Hogema et al., 2012). Chronic Q fever symptoms may be endocarditis, chronic
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199 fatigue syndrome and pregnancy difficulties. Endocarditis is the major form of chronic Q
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200 fever, comprising 60%–78% of all cases worldwide and can occur months, even years after

201 an acute infection (Arricau-Bouvery & Rodolakis, 2005). Predisposing conditions such as

202 pre-existing valvular disease, vascular abnormalities and immunosuppression increase the

203 risk of endocarditis (Karakousis & Trucksis, 2006).

204 Q-fever fatigue syndrome can also occur after an acute Q fever infection. It has been

205 described worldwide in up to 20%-30% of patients and can last for months or years
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206 (Arricau-Bouvery & Rodolakis 2005). Q-fever fatigue syndrome is also followed with

207 symptoms such as night sweats, myalgia, arthralgia, mood swings and changes in

208 sleeping pattern (Arricau-Bouvery & Rodolakis, 2005).

209 A lot of parameters can affect the clinical polymorphism of Q fever and major

210 differences in the manifestations of disease in various countries have been observed. For

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211 example, pneumonia is the major clinical presentation in Nova Scotia, Canada, in the

212 Basque region in Spain, in Switzerland, in Greece, in Kroatia and Maritime Canada

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213 (Alexiou-Daniil et al., 1990; Raoult et al., 2000; Lukšić, Punda-Polić, Ivić, Bradarić, &

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214 Bradarić 2006). Hepatits is mostly common in Ontario, Canada, in Andalusia in Spain, and

215 in California, USA (Raoult et al., 2000). In Australia, fever with no apparent localization of

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216 infection predominates; and in France, southern Spain and the Canary Islands, the
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217 predominant symptoms are fever and hepatitis (Parker & Barralet 2006; Marrie &

218 Campbell 2008).

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219 Q fever infections during pregnancy are almost always asymptomatic (Tissot-Dupont
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220 et al., 2007). However, the symptoms in infected pregnant women may be fever, flu-like
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221 illness, severe thrombocytopenia and atypical pneumonia (Maurin & Raoult 1999).

222 Transplacental infection of the fetus in utero is possible, but its consequences are still
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223 unknown (Carcopino, Raoult, Bretelle, Boubli, & Stein, 2007). Among Q fever pregnancy

224 problems there are placentitis, immediate abortion, fetus abnormalities, premature
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225 delivery and low birth weight (Carcopino et al., 2007). Q fever may also lead to repeated
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226 abortions in several pregnancies (Arricau-Bouvery & Rodolakis 2005). However, recent

227 studies quantifying the consequences of an infection with C. burnetii during pregnancy

228 did not show any evidence of adverse pregnancy outcome at the women, living in regions

229 with the highest incidence levels of Q fever (Nielsen et al., 2013).

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231 C. burnetii in milk and its role in human infection

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232 Among food products of animal origin, the raw milk is considered as the most

233 significant source of C. burnetii (Capuano et al., 2012; Hilbert et al., 2015). C. burnetii is

234 excreted in the milk of infected animals (cattle, sheep and goats) with clinical signs of

235 infection or not for variable periods during lactation. In an early study, Enright, Sadler,

236 and Thomas (1957) showed that infected cattle can shed viable C. burnetii in milk for a

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237 time longer than 1 year. Guatteo, Joly, and Beaudeau (2012) identified three infected cows

238 as persistent shedders of the pathogen at high population levels of the pathogen in the

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239 milk at 14, 21 and 28 days post-abortion. C. burnetii DNA was identified in milk of infected

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240 goats up to 38 days (Roest et al., 2012) and 52 days (Arricau-Bouvery, Souriau, Lechopier,

241 & Rodolakis, 2003) post-partum.

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242 Faecal materials can contaminate the milk of lactating animals (EFSA, 2010). C.
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243 burnetii has spore-like stability in the environment due to SCV morphotype which most

244 likely exists in the milk and records for the long also the stability of the pathogen. In fact,
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245 C. burnetii does not live outside the intracellular environment of the host cells. In this
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246 manner, for risk evaluation, it is accepted that the development of the pathogen in the
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247 milk and dairy items does not happen (Gale et al., 2015).

248 Consumption of raw milk and raw milk products possess a relatively higher risk of C.
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249 burnetii infection as compared to the consumption of both pasteurized milk and

250 pasteurized milk products. In fact, Enright et al. (1957) reported that heating milk at 145
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251 F/62.77 oC for 30 min inactivated C. burnetii in concentrations of 100,000 infective guinea
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252 pig doses per 2 ml. Further studies demonstrated that the current official standard of milk

253 pasteurization, 161 οF/71.66 oC for 15 s is effective in eliminating C. burnetii (EFSA, 2010).

254 Pasteurization is designed to achieve at least a 5-log reduction of C. burnetii in whole milk

255 (Hudson, Wong, & Lake, 2003). Studies conducted to ascertain the thermal resistance of

256 C. burnetii showed a D value of 0.5-0.6 min at 65.6 oC and z values of 4.4-5.5 oC (Juff &

257 Deeth, 2007).

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258 The presence of C. burnetii in milk raises concern on the role of raw milk as a human

259 infection source, particularly in regions where unpasteurized milk is consumed (Loftis,

260 Priestley, & Massung, 2010). After the Second World War, a high prevalence of Q fever

261 was observed among the population in European and North American regions with a high

262 raw milk and raw milk products consumption (Wegener, 1957; Marmion & Stoker 1958).

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263 Beck and Bell (1949) reported that 300 Q fever cases in Los Angeles were associated with

264 the consumption of raw milk. An early epidemiological study of Q fever cases in the

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265 United Kingdom in 1984 to 1994, showed that three cases were caused by consuming

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266 unpasteurized milk (Pebody, Wall, Ryan, & Fairley, 1996). In France, an outbreak of Q

267 fever with 61 cases among patients and staff of a psychiatric institution was also

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268 associated with the consumption of raw milk (Fishbein & Raoult, 1992). Similar cases of Q
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269 fever after raw milk consumption were also reported in various countries (Brown,

270 Colwell, & Hooper, 1968; Brouqui et al., 1993; Serbezov et al., 1999). Recent surveys
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271 indicate that the consumption of raw milk and/or raw milk products contaminated with C.
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272 burnetii has been associated with sero-prevalence in humans (Hatchette et al. 2001;
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273 Maltezou et al. 2004).

274 Due to the high number of target macrophages in the lungs, C. burnetii can easier
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275 infect the respiratory system than the digestive one (Gale et al. 2015). C. burnetii

276 originated from the birth product tissues may be more infectious through certain routes
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277 (e.g. intraperitoneal challenge) as compared to this of the consumption of contaminated

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278 raw milk (Gale et al. 2015).

279 Experiments involving human volunteers who consumed raw milk have generated

280 contradictory results. In a study conducted by Benson et al. (1963), the serological

281 analysis showed that individuals consumed raw milk contaminated with pathogen had a

282 higher C. burnetii seroprevalence than individuals in control groups, but no-one showed

283 any sign of clinical disease. In an asylum in Portugal, contaminated food with C. burnetii
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284 consumed by 11 persons, resulted in a seropositive presence by complement fixation

285 assay in only two persons, but none of them developed clinical symptoms (Fonseca et al.

286 1949). Similar seroconversion findings with no any infection or clinical signs were also

287 reported by Fishbein and Raoult (1992). Krumbiegel and Wisniewski (1970) reported no

288 findings of Q fever infection in a group of 34 human volunteers, after the consumption of

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289 contaminated raw milk. The authors attributed their findings to the dosage and Coxiella

290 strains in the contaminated milk. However, goat workers consumed cheese made from

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291 contaminated raw caprine milk in Newfoundland (Hatchette et al., 2001) and children

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292 consumed cheese made by contaminated raw milk in Greece developed Q fever

293 (Maltezou et al., 2004). According to a recent study in 2011, consumption of

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294 contaminated raw cow’s milk from the same dairy in Michigan lead 5 individuals to
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295 infection with C. burnetii (Signs, Stobierski, & Gandhi, 2012).

296
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297 Detection of C. burnetii in milk

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298 The polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA)
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299 methods are the most common methods used to identify C. burnetii presence in raw milk.

300 These methods are also used for either individual milk samples (per animal) or bulk tank
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301 milk samples (BTM) in several surveys conducted in various countries for the C. burnetii to

302 highlight the C. burnetii presence. The same methods are also commonly employed to
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303 identify C. burnetii infections in cattle, sheep and goat flocks.

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304 Several PCR techniques such as conventional PCR, real-time PCR, multiplex PCR and

305 nested PCR are usually used to detect the presence of C. burnetii DNA in milk samples

306 (van den Brom, van Engelen, Roest, van der Hoek, & Vellema 2015). The PCR assay is able

307 to identify C. burnetiii in milk immediately after the contamination, while serologic assays

308 can identify antibodies which developed in milk after a longer period of time. Real-time

309 PCR in milk samples is now commonly used to support a diagnosis of C. burnetii
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310 abortion/stillbirth in animals (Horigan, Bell, Pollard, Sayers, & Pritchard, 2011). Both SCVs

311 and LCVs forms of C. burnetii DNA in milk can also be detected by PCR assay. The SCV

312 represents a higher exposure risk to human health because this form can survive not only

313 in the milk but also in the human intestine (Gale et al. 2015).

314 Examination of BTM samples by using PCR tests for the identification of infected

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315 animals flocks has some limitations. Sampling shortly after lambing might lead to higher

316 prevalence (Rodolakis et al., 2007). A positive result can be derived by only a few

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317 shedding animals at the time of sampling. Findings of C. burnetii DNA in milk do not

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318 always coincide with serological finding since the seronegative animals can also excrete

319 C. burnetii in milk (Rousset et al., 2009). A BTM PCR negative result does not reveal the

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320 existence of infected animals, since they may shed C. burnetii via other routes. With
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321 reference to this, the use of repeated sampling can reduce a likelihood of false

322 conclusions (Guatteo, Beaudeau, Joly, & Seegers, 2007).

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323 The fundamental drawback of PCR procedures was that a positive outcome was not
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324 really a sign that the milk contains infective C. burnetii cells (Vellema, van den Brom,
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325 Dercksen, Moll, & Roest, 2010). Loftis et al. (2010) have confirmed the viability by isolation

326 of the agent in tissue culture in two PCR-positive available on the market, unpasteurized
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327 milk samples. Recently, a PCR methodology (ethidium monoazide-PCR) has been

328 published for C. burnetii that circumvents this issue due to its capacity to differentiate the
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329 live and dead cells (Mori et al., 2013).

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330 ELISA tests are widely used to detect C. burnetii antibodies in milk. ELISA is a cost-

331 effective and moderately great index of the seroprevalence of C. burnetii in various

332 animals but cannot identify shedders in contrast to quantitative real-time PCR (Czaplicki

333 et al., 2012). The detection of antibodies by ELISA in milk seems more sensitive than it is in

334 serum (Rodolakis et al., 2007). However, ELISA assays may over-estimate prevalence in

335 animals with no C. burnetii cells present but seropositive for life (Gale et al., 2015).
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336

337 Prevalence of C. burnetii in milk

338 Accomplished data on the prevalence of C. burnetii in milk samples by employing PCR

339 from published surveys conducted in various countries are summarized in Table 1. The

340 majority of these surveys were conducted in Europe; it may be due to the large outbreak

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341 of C. burnetii recorded in the Netherlands in 2007–2009 (Vanderburg et al., 2014).

342 - Europe

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343 In Belgium, Czaplicki et al. (2012) found a C. burnetii prevalence of 30% in BTM bovine

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344 milk tested by real time PCR analysis during a survey conducted in 2006. In Belgium, a

345 survey conducted in BTM samples from dairy goat farms during December 2009 to March

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346 2013 showed a prevalence of C. burnetii ranging from 6.3 to 12.1% (Boarbi et al., 2014). In
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347 France, Rodolakis et al. (2007) conducted a survey on 3 herds by examining milk samples

348 of 95 cows, 120 goats, and 90 ewes over 16 weeks and reported a prevalence of C.
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349 burnetii of 24%, 19% and 16%, respectively. In France, Guatteo et al. (2007) examined milk
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350 samples of infected cows by using real time PCR and reported that the percentage of
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351 positive samples was 24.4%. A BTM survey carried out in south-west England showed a

352 prevalence of 69.7% in dairy cattle (Valergakis, Russell, Grogogno-Thomas, Bradley, &
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353 Eisler, 2012).

354 In the Netherlands, van den Brom et al. (2012) examined BTM samples of caprine and
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355 ovine farms in 2008 and found a C. burnetii prevalence of 32.9% and 0%, respectively. In
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356 the same country, Muskens, van Engelen, van Maanen, Bartels, and Lam (2011) reported a

357 C. burnetii prevalence of 8.7% among milk samples of lactating cows. In Switzerland,

358 Fretz, Schaeren, Tanner, and Baumgartner (2007) examined the incoming bovine, ovine

359 and caprine milk in two cheese plants for the presence C. burnetii by using nested PCR.

360 The workers reported a C. burnetii prevalence of 4.7% in the BTM bovine milk, while all the

361 ovine and caprine bulk milk samples were found negative. In Switzerland, a survey carried
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362 out in bovine BTM showed the presence of C. burnetii DNA in 29.4% of the analyzed

363 samples (Baumgartner, Niederhauser, & Schaeren, 2001). In Denmark, Angen, Ståhl,

364 Agerholm, Christoffersen, and Agger (2011) examined 1,514 BTM samples of bovine herds

365 and found a C. burnetii prevalence of 32%.

366 In Italy, Petruzzelli et al. (2013) examined 130 BTM bovine milk samples from 14 farms

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367 and reported a C. burnetii prevalence of 27%. In Italy, surveys carried out by Magnino et al.

368 (2009) and Valla, Bizzarri, Ferrari, and Bussacchini (2014) in bovine BTM samples revealed

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369 almost the same C. burnetii prevalence of 40% and 40.10%, respectively. Similarly, Vicari et

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370 al. (2013) examined BTM bovine milk in northern Italy and found a C. burnetii prevalence

371 of 43%.

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372 In Spain, Astobiza et al. (2012) reported a C. burnetii prevalence of 51.7% in bovine BTM
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373 samples by PCR analysis. In northern Spain, a survey carried out on ovine BTM samples

374 revealed a C. burnetii prevalence of 22% (García-Pérez et al., 2009). In Portugal, Anastácio,
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375 Carolino, Sidi-Boumedine, and da Silva (2016) conducted a survey on BTM samples from
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376 cattle and small ruminant dairy herds and found a C. burnetii prevalence of 20% and 6.3%,
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377 respectively. In Hungary, C. burnetii DNA was detected by real-time PCR in 8.7% of

378 individual cow’s milk samples, 4.0% of individual ewe’s milk samples, and 66.7% of bovine
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379 BTM samples (Gyuranecz et al., 2012). In Germany, Hilbert et al. (2015) detected C. burnetii

380 DNA in 62.5% of the bovine BTM samples and up to 60% of individual cow’s milk samples.
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381 samples, and 6.2% (Rahimi et al., 2010) or 8.3% (Haghi et al., 2015) in bovine BTM milk
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382 samples. In China, C. burnetii DNA was detected by real-time PCR (FRET-qPCR) in 1.3% of

383 bovine BTM milk samples (El-Mahallawy et al., 2016).

384 - America

385 In the USA, official surveys on BTM samples from 528 dairy farms in 18 states during

386 March – August 2007 showed a C. burnetii prevalence of 76.9 % (APHIS, 2007). In the USA,

387 Kim, Kim, Lafferty, and Dubovi (2005) conducted a survey (January 2001 to December
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388 2003), and observed a C. burnetii prevalence of 94% in BTM samples from US dairy herds,

389 which is the highest one recorded in the world. In the USA (Indiana), the prevalence of C.

390 burnetii was 60.08% and 2.50% in bovine (Bauer et al. 2015) and caprine (Bauer et al. 2016)

391 BTM samples, respectively. In the USA, C. burnetii DNA was detected in 9 out of 21 (42.9%)

392 raw bovine milk samples obtained in seven states (Loftis et al. 2010). In Colombia,

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393 Contreras, Máttar, González, Álvarez, and Oteo (2015) reported the presence of C.

394 burnetii DNA in 45% of BTM raw bovine samples.

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395 - Asia

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396 In Turkey, Can, Elmali, and Karagöz (2015) reported a C. burnetii prevalence of 10% in

397 cows’ bulk milk and 4% in goats’ or ewes’ bulk milk samples. In Turkey, according to a

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398 survey conducted by Gulmez Saglam and Sahin (2016) the C. burnetii prevalence was 1.42%
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399 in bovine BTM and 0.4% in ovine BTM milk samples. In Iran, surveys conducted in various

400 provinces revealed C. burnetii prevalence of 0% (Rahimi, Doosti, Ameri, Kabiri, & Sharifian,
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401 2010) and 3.3% (Haghi et al., 2015) in ovine BTM milk samples, 1.8% (Rahimi et al., 2010), 2%
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402 (Rahimi 2010) and 16.12% (Khalili, Diali, Mirza, & Mosavi, 2015) in caprine BTM milk
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403 samples, and 6.2% (Rahimi et al., 2010) or 8.3% (Haghi et al., 2015) in bovine BTM milk

404 samples. In China, C. burnetii DNA was detected by real-time PCR (FRET-qPCR) in 1.3% of
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405 bovine BTM milk samples (El-Mahallawy et al., 2016).

406 - Africa
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407 In Africa the only published survey was conducted in Gambia, and C. burnetii DNA was
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408 found in 2 out of 67 caprine milk samples (Klaasen et al., 2014).

409 - Oceania

410 It is important to note that there are no surveys available on C. burnetii in milk in

411 countries of Oceania, since recent studies report a zero prevalence of C. burnetii in

412 domestic ruminants in New Zealand (Guatteo et al., 2012).

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413 Table 2 shows accomplished data on the prevalence of antibodies to C. burnetii in milk

414 samples by employing ELISA from published studies conducted in various countries.

415 -Europe

416 In 2006, a survey on bovine BTM samples was carried out in southern Belgium and

417 57.8% of herds were found seropositive (Czaplicki et al., 2012). Agger, Christoffersen,

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418 Rattenborg, Nielsen, and Agerholn (2010) based on ΒΤΜ samples ELISA examination

419 reported a C. burnetii positivity rate of 59% in randomly selected cattle herds in Denmark.

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420 In Denmark, Angen et al. (2011) reported that 25% of 1,514 individual milk samples of

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421 Danish dairy herds were positive in the presence of C. burnetii antibodies. In Ireland, 37.9%

422 of bovine BTM samples of dairy herds were antibody positive in the pathogen (Ryan et

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423 al., 2011). In the Netherlands, 29.8% and 18.8% of BTM samples from sheep and goat farms
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424 were ELISA positive in C. burnetii, respectively (van den Brom et al., 2012). Muskens et al.

425 (2011) reported that infection with the pathogen was widespread among Dutch dairy
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426 herds, since anti-C. burnetii antibodies were detected in 78.6% of BTM bovine milk
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427 samples.
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428 In France, Guatteo et al. (2007) detected the presence of anti-C. burnetii antibodies in

429 57.36% of individual cow’s milk samples. In England and Wales, Paiba, Green, Lloyd, Patel,
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430 and Morgan (1999) conducted a survey on BTM samples from dairy cattle farms by using

431 ELISA test and reported a prevalence of C. burnetii of 21%. In Spain, 66.9% of bovine BTM
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432 milk samples were positive for the presence of C. burnetii antibodies (Astobiza et al.,
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433 2012). In Spain, a survey on ovine BTM milk samples revealed the presence of antibodies

434 against C. burnetii in 40.3% of the examined samples (Ruiz-Fons et al., 2011). In Portugal,

435 Anastácio et al. (2016) examined BTM milk samples from cattle and small ruminant herds

436 by ELISA and estimated a C. burnetii prevalence of 37.8% and 51.6%, respectively. In

437 Greece, bovine BTM bulk milk samples from dairy cattle herds assayed by ELISA for C.
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438 burnetii antibodies showed a prevalence of 35% (Dovolou, Tsiligianni, Vouzaras, & Amiridis

439 2011).

440 - Asia

441 In Iran, Khalili, Sakhaee, Aflatoonian, and Shahabi-Nejad (2011) conducted a survey on

442 BTM bovine milk samples from farms in the southeast Iranian region by using ELISA test

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443 and found a prevalence of 45.4%. In Turkey, 10.28% of cow’s and 16.8% of sheep BTM milk

444 samples examined by ELISA were seropositive (Gulmez Saglam & Sahin, 2016).

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445 According to the accomplished surveys on the presence of C. burnetii in milk by

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446 employing PCR or ELISA tests, the pathogen is present in the bovine, ovine or caprine

447 milk in several countries of the world, and the bovine milk shows higher prevalence

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448 records than those of ovine or caprine milk.
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449

450 Conclusions
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451 Q fever is an important zoonosis. The Q fever in human is characterized by clinical

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452 polymorphism and may be present as acute Q fever or chronic Q fever. After the initial
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453 infection, an almost half of the patients are asymptomatic. In the last decades, Q fever

454 outbreaks have been reported in various countries, indicating its importance as an
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455 emerging public health threat. According to surveys conducted in many countries, the

456 prevalence of C. burnetii in raw milk samples has been varied from 0% to as high as 95%.
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457 The highest records of prevalence of the pathogen were observed in bovine milk as
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458 compared to those in milk of small ruminants. Based on current data, the risk of C.

459 burnetii infection by consuming unpasteurized milk and raw milk dairy products may be

460 not negligible. Further studies on the epidemiological effects of the C. burnetii presence

461 in raw milk and Q fever onset are required.

462

463 Funding resource

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464 This research did not receive any specific grant from funding agencies in the

465 public, commercial, or not-for-profit sectors.

466

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467 References

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663 Coxiella burnetii (Q fever) in bulk tank milk in England and Wales. Veterinary Record, 144, 519-522.
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664 Pape M., Bouzalas E. G., Koptopoulos G. S., Mandraveli K., Arvanitidou-Vagiona M., Nikolaidis P., &

665 Alexiou-Daniel St. (2009). The serological prevalence of Coxiella burnetii antibodies in sheep and

666 goats in northern Greece. Clinical Microbiology and Infection,15,46-147.

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676 Rahimi E, Ameri M, Karim G, Doosti A. (2011). Prevalence of Coxiella burnetii in bulk milk samples

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688 Coxiella burnetii Shedding in Milk of Dairy Bovine, Caprine, and Ovine Herds. Journal of Dairy

689 Science, 90,5352–5360.

690 Roest, H-J., van Gelderen B., Dinkla A., Frangoulidis D., van Zijderveld F., Rebel J., & van Keulen L.

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694 shedding routes and antibody response after outbreaks of Q fever-induced abortion in dairy goat

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703 Bulgaria and Slovakia. Emerging Infectious Disease, 5,388–394.

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713 Infectious Diseases, 44, 232–237.

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714 Valergakis G. E., Russell C., Grogogno-Thomas R., Bradley A. J., & Eisler M. C. (2012). Coxiella
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715 burnetii in bulk tank milk of dairy cattle in south-west England. Veterinary Record, 171,156.

716 Valla G., Bizzarri D., Ferrari G., &Bussacchini M. (2014). Prevalence of Coxiella burnetii in bulk milk in

717 herds of dairy cows and possible correlation with Italian reproductive problems. Large Animal

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719 van den Brom R., van Engelen E., Luttikholt S., Moll L., van Maanen K., & Vellema P. (2012). Coxiella

720 burnetii in bulk tank milk samples from dairy goat and dairy sheep farms in The Netherlands in

721 2008. Veterinary Record, 170,310.

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724 Vanderburg S., Rubach M. P., Halliday J. E., Cleaveland S., Reddy E. A., &Crump J. A. (2014).

725 Epidemiology of Coxiella burnetii Infection in Africa: A One Health Systematic Review.PLoS

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726 Neglected Tropical Diseases, 8, e2787.

727 Vellema P., van den Brom R., Dercksen D. P., Moll L., & Roest H. J. (2010). Research in relation to

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728 the approach of Q fever in the Netherlands. Q-fever conference, Breda, the Netherlands, February

729 2010.

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730 Vellema P., & van den Brom R. (2014). The rise and control of the 2007–2012 human Q fever
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731 outbreaks in the Netherlands. Small Ruminant Research, 118, 69–78.

732 Vicari N., Faccini S., Ricchi M., Garbarino C., Decastelli L.., Boldini M, et al. (2013). Occurrence of
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733 Coxiella burnetii in bulk tank milk from northwestern Italy. Veterinary Record, 172,687.

734 Wegener K. H. (1957). Q Fever and its signification regarding milk hygiene. Bibliographic study with
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735 contribution to the question of its occurrence in cattle herds in Schleswig-Holstein. Kieler
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736 Milchwirtschaftliche Forschungsberichte, 9, 509–535.

737
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738

739
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740
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741

742

743

744

745

746

747
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748 Table 1

749 Prevalence of C. burnetii in raw milk by employing PCR in published studies around the

750 world.

PT
RI
Year Type of
Number %
Country of milk Test Reference

SC
tested positive
study sample

EUROPE

Bovine
U real-time
AN
Belgium 2006 50 30 Czaplicki et al., 2012
BTMa PCR

2009- Caprine real-time

M

Belgium 1,924 6.3-12.1 Boarbi et al., 2014

2013 BTM PCR
D

Bovine

Denmark 2008 milk 1,514 32 PCR Angen et al., 2011

TE

samples

2009- Bovine real-time

EP

England 155 69.7 Valergakis et al., 2012

2010 BTM PCR
C

Bovine
France - 95 24 PCR Rodolakis et al., 2007
BTM
AC

Caprine
France - 120 16 PCR Rodolakis et al., 2007
BTM

Ovine
France - 90 19 PCR Rodolakis et al., 2007
BTM

Bovine

Germany - bulk milk 173 62.5 PCR Hilbert et al., 2015

samples

Bovine
Germany - 2,807 60 PCR Hilbert et al., 2015
milk
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samples

Bovine
real-time
Hungary - milk 150 8.7 Gyuranecz et al., 2012
PCR
samples

Bovine real-time
Hungary - 15 66.7 Gyuranecz et al., 2012

PT
BTM PCR

Ovine milk real-time

Hungary - 50 4.0 Gyuranecz et al., 2012

RI
samples PCR

2007- Bovine

SC
Italy 400 40 PCR Magnino et al., 2009
2008 BTM

Bovine

U
Italy - 130 27 PCR Petruzzelli et al., 2013
BTM
AN
2007- Bovine
Italy 780 43 PCR Vicari et al., 2013
2011 BTM
M

2011- Bovine
Italy 344 40.1 PCR Valla et al., 2014
2013 BTM
D

Bovine real-time
TE

Netherlands 2007 341 56.6 Muskens et al., 2011

BTM PCR

Bovine
EP

real-time
Netherlands 2008 milk 2,925 8.7 Muskens et al., 2011
PCR
samples
C

Caprine real-time van den Brom et al.,

AC

Netherlands 2008 292 32.9

BTM PCR 2012

Ovine real-time van den Brom et al.,

Netherlands 2008 16 0
BTM PCR 2012

Bovine
Portugal - 45 20 PCR Anastácio et al., 2016
BTM

Portugal - Small 64 6.3 PCR Anastácio et al., (2016)

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ruminants

BTM

Ovine
Spain - 154 22 PCR García-Pérez et al., 2009
BTM

2009- Bovine
Spain 178 51.7 PCR Astobiza et al., 2012

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2010 BTM

Bovine nested
Switzerland 2006 359 4.7 Fretz et al., 2007

RI
bulk milk PCR

Ovine nested

SC
Switzerland 2006 81 0 Fretz et al., 2007
bulk milk PCR

Caprine nested

U
Switzerland 2006 39 0 Fretz et al., 2007
bulk milk PCR
AN
Bovine quantita
Switzerland 2007 872 39.4 Baumgartner et al., 2011
BTM tive PCR
M

ASIA
D

real-time
Bovine
(FRET-
TE

China - milk 150 1.3 El-Mahallawy et al., 2016

qPCR)
samples
PCR
EP

Caprine
nested
C

Iran - milk 296 2.0 Rahimi 2010

PCR
sample
AC

Bovine nested
Iran 2008 210 6.2 Rahimi et al., 2010
BTM PCR

Ovine nested
Iran 2008 110 0 Rahimi et al., 2010
BTM PCR

Caprine nested
Iran 2008 56 1.0 Rahimi et al., 2010
BTM PCR
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Bovine nested
Iran - 247 3.2 Rahimi et al., 2011
bulk milk PCR

Ovine nested
Iran - 140 5.7 Rahimi et al., 2011
bulk milk PCR

Caprine nested
Iran - 110 4.5 Rahimi et al., 2011

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BTM PCR

Caprine
nested

RI
Iran - milk 296 2.0
PCR Abbasi et al., 2011
samples

SC
Bovine nested
Iran - 100 11 Kargar et al., 2013
BTM PCR

Caprine

U trans-
AN
Iran 2012 31 16.12 Khalili et al., 2015
bulk milk PCR

Bovine
M

38
Iran 2014 milk 8.3 PCR Haghi et al., 2015

samples
D

Ovine milk
TE

Iran 2014 22 3.3 PCR Haghi et al., 2015

samples

Caprine
EP

nested
Iran 2013 milk 72 20.83 Lorestani et al., 2016
PCR
samples
C

2012- Bovine
AC

Turkey 50 10 PCR Can et al., 2015

2013 bulk milk

Caprine
Turkey 50 4 PCR Can et al., 2015
bulk milk

Ovine
Turkey 50 4 PCR Can et al., 2015
bulk milk

Turkey - Bovine 350 1.42 trans- Gulmez Saglam &Sahin,

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milk PCR 2016

trans- Gulmez Saglam &Sahin,

Turkey - Ovine milk 250 0.4
PCR 2016

AMERICA

Bovine
Colombia 2012 11 45 PCR Contreras et al., 2015

PT
BTM

trans-

RI
2001- Bovine
USA 316 94.3 PCR Kim et al., 2005
2003 BMT

SC
Bovine
USA 2007 528 76.9 PCR APHIS, 2007
BMT

Bovine/
U
AN
14
USA Caprine
- bovine 42.9 PCR Loftis et al., 2010
(12 states) milk
M

7 caprine
samples
D

Bovine real time

USA (Indiana) 2011 316 60.8 Bauer et al., 2015
BMT PCR
TE

2012- Caprine real-time

USA (Indiana) 387 2.5 Bauer et al., 2016
2014 milk PCR
EP

AFRICA
C

Caprine
AC

Gambia 2012 Milk 33 2.94 PCR Klaasen et al., 2014

samples

Ovine Milk
Gambia 2012 34 3.03 PCR Klaasen et al., 2014
samples

a
751 BTM: bulk tank milk
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752 Table 2

753 Prevalence of C. burnetii in raw milk by employing ELISA in published studies around the

754 world.

Type of
Year of Number %
Country milk Test Reference

PT
study tested positive
sample

RI
EUROPE

Bovine indirect

SC
Belgium 2006 206 57.8 Czaplicki et al., 2012
a
BTM ELISA

2009- Caprine

U
Belgium 1924 6.3 -12.1 ELISA Boarbi et al., 2014
2013 BTM
AN
Bovine Bodker &
Denmark 2007 742 57 ELISA
BTM Christoffersen, 2008
M

Bovine
Denmark 2008 100 59 ELISA Agger et al., 2010
BTM
D

Bovine
TE

Denmark 2008 milk 1,514 25 ELISA Angen et al., 2011

samples
EP

England and Bovine

- 373 21.2 ELISA Paiba et al., 1999
Wales BTM
C

Bovine
AC

France - milk 145 40 ELISA Guatteo et al., 2007

samples

Bovine
Greece - 80 35 ELISA Dovolou et al., 2011
bulk milk

Bovine
Ireland 2009 290 37.9 ELISA Ryan et al., 2011
bulk milk
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Bovine
Netherlands 2007 341 78.6 ELISA Muskens et al., 2011
BTM

Caprine van den Brom et al.,

Netherlands 2008 292 29.8 ELISA
BTM 2012

Ovine van den Brom et al.,

Netherlands 2008 16 18.8 ELISA

PT
BTM 2012

Bovine
Portugal - 45 37.8 ELISA Anastácio et al., 2016

RI
BTM

Small

SC
Portugal - ruminant 64 51.6 ELISA Anastácio et al., 2016

s BTM

Ovine

U indirect
AN
Spain 2005 154 40.3 Ruiz-Fons et al., 2011
BTM ELISA

2009- Bovine indirect

M

Spain 178 66.9 Astobiza et al., 2012

2010 BTM ELISA
D

ASIA

Bovine
TE

Iran - 44 45.4 ELISA Khalili et al., 2011

BTM

Bovine
EP

Gulmez Saglam &

Turkey - milk 350 10.28 ELISA
Sahin 2016
C

samples

Ovine
AC

Gulmez Saglam &

Turkey - milk 250 16.8 ELISA
Sahin, 2016
samples

a
755 BTM: bulk tank milk.

756
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Highlights

• Characteristics of C. burnetii the causative agent of Q fever are presented.

• Infection sources and epidemiology of Q fever are discussed.
• The presence of C. burnetii in milk and its role in human infection is
reviewed.
• Detection methods of C. burnetii in milk are presented.

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• The worldwide prevalence of C. burnetii in milk samples is presented.

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