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Q fever, state of art: Epidemiology, diagnosis and prophylaxis
Small Ruminant Research 62 (2006) 121–124
Q fever, state of art: Epidemiology, diagnosis and prophylaxis夽 A. Rodolakis ∗ INRA, Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France Available online 22 September 2005
Abstract Q fever is a widespread zoonosis caused by Coxiella burnetii. The respiratory tract is the most common route of infection, which occurs by inhalation of contaminated dust and spray shed from infected animals. Livestock is considered as the major “source” for human infections. This short review summarizes the state of the knowledge of the diagnosis, treatment and prevention of Q fever in sheep flocks. © 2005 Elsevier B.V. All rights reserved. Keywords: Sheep; Abortion; Coxiella burnetii; Vaccination; Control
1. Introduction Q fever (for query fever), a zoonosis caused by the obligate intracellular microorganism Coxiella burnetii, is endemic throughout the world and infects arthropods, birds, pets, domestic and wild mammals, as well as humans. The disease is known since the 1930s and has a worldwide distribution, with the exception of the Antarctica and possibly New Zealand. In ewes, C. burnetii infections are generally asymptomatic, but they can lead to abortions, stillbirths and delivery of weak and unviable lambs. In the majority of cases, abortions occur at the end of gestation without specific clinical signs until abortion is imminent. 夽 This paper is part of a special issue entitled Keynote Lectures of
the 6th International Sheep Veterinary Congress—Guest Edited by Dr. George C. Fthenakis and Prof. Quintin A. McKellar. ∗ Tel.: +33 24741 7700; fax: +33 247 427 7779. E-mail address: [email protected]. 0921-4488/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2005.07.038
Such reproductive failures are accompanied with shedding of great number of Coxiella into birth products, urine, faeces and milk of infected animals. Aborted foetuses appear normal, but infected placentas exhibit intercotyledonary fibrous thickening and discolored exudates, which are not specific to Q fever. The abortion rate is generally low. It can range from 3 to 80% of pregnant female, but the higher abortion percentages are rarely observed, except in some goat herds. In sheep flocks, the number of females, which abort, may not be enough to alert the farmer and human clinical cases often reveal the ovine infection. In humans, the acute disease appears like a flu-like infection, usually self-limiting illness accompanied by myalgia and severe headache. Complications, such as pneumonia or hepatitis, may also occur. Endocarditis in patients suffering from valvulopathy, as well as premature delivery or abortion in pregnant women, are the main severe manifestations of the chronic evolution of the disease.
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A. Rodolakis / Small Ruminant Research 62 (2006) 121–124
The source of human infection is often unidentified, although sheep and goats are more frequently involved in the disease cycle than other animal species. The main route of C. burnetii infection is by inhalation of contaminated aerosols or dusts containing the microorganism shed from infected animals. As C. burnetii is very stable in the environment, resisting to elevated temperature, desiccation, osmotic shock, ultra-violet light and disinfectants, direct contact with an aborting female is not required. People may be infected by handling contaminated wool, manure or clothes contaminated with feces or by the transhumance of infected flocks through a valley. The airborne transmission of C. burnetii associated with its resistance and the ability of producing easily a large quantity of organisms in the placenta of an aborting ewe or doe, have contributed to consider C. burnetii as a category B biological terrorist agent, although the great number of asymptomatic infections limits the consequence of its use as biological weapon. Oral transmission, by ingestion of contaminated raw milk or dairy products in particular goat dairy products could lead to seroconversion and in few cases to Q fever.
2. Antigenic variations of C. burnetii C. burnetii is unique among rickettsiae in that its lipopolysaccharide (LPS) presents a phase variation, similar to smooth-rough variation in the LPS of enterobacteria. Virulent C. burnetii designed phase I corresponds to “smooth-type” LPS and is infectious for animals and humans contrary to phase II C. burnetii obtained after several passages in chicken embryos or cells culture. Phase II C. burnetii is more readily phagocytosed than is phase I C. burnetii, but it is killed by complement contrary to phase I organisms, which resist to complement-mediated serum killing. Phase II C. burnetii has a truncated LPS typical of “rough” LPS: the carbohydrate portion of phase II LPS lacks many of the sugars present in phase I LPS. In addition to phases I–II LPS antigenic variation, other antigenic variations in LPS (Hackstadt, 1986) and in outer membrane proteins (To et al., 1998) of C. burnetii isolates from various sources have been demonstrated. In particular antigenic variations have been observed between C. burnetii strains isolated from ruminants and the reference Nine Mile C. burnetii
strain isolated from a tick in USA; that could explain some lack of sensibility of ELISA kits using Nine Mile C. burnetii strain as antigen.
3. Diagnosis Since there are no specific signs of Q fever, suitable laboratory tests are required for accurate diagnosis. The diagnosis of Q fever remains difficult and epidemiological studies are often based only on serological investigations, which have the potential disadvantage of indicating exposure rather than the detection of the organism. Routine diagnosis of Q fever is usually established by examination of fixed impressions or smears prepared from the placenta stained by the Stamp, Gimenez or Machiavello methods, associated with serological tests. Isolation of C. burnetii is not performed for routine diagnosis in veterinary medicine, because C. burnetti does not grow on standard laboratory bacteriological media and its isolation takes long time and is difficult and hazardous to perform: it requires confined level 3 laboratories (L3). Therefore, isolation of C. burnetii is restricted to specialized laboratories by using the shell-vial cell culture technique (Raoult et al., 1990) or culture on chicken embryos. Recently, polymerase chain reaction (PCR) has radically changed the diagnosis of Q fever in veterinary medicine (Berri et al., 2003). It is the only one, which allows identifying shedders. PCR kits are becoming available and provide a specific, sensitive and rapid tool for the detection of C. burnetii in various clinical samples. The CF test, which is the OIE prescribed serological test, is weakly sensitive and the antigen used in this test frequently fails to detect antibodies in sheep or goats (Kovacova et al., 1998). Moreover, the cutoff value used for diagnosis of chlamydiosis is often used for Q fever, leading to mistakes. The speciesspecific indirect immunofluorescence assay is not often used for diagnosis of Q fever in animals. The ELISA test is more sensitive than the CF test and allows for testing a greater number of animals and flocks. However, this does not allow individual identification of animals that shed C. burnetii in faeces or milk. If most animals, which excrete C. burnetii in vaginal mucus, faeces or milk, are seropositive, some animals can be seropositive without shedding C. burnetii and a few
A. Rodolakis / Small Ruminant Research 62 (2006) 121–124
ones can excrete and remain seronegative (Berri et al., 2002). This last situation has an important consequence for animal and public health, and is due to the antigen used in this test instead of a lack of antibodies response as we have demonstrated it by using ELISA or immunofluorescence assay with various C. burnetii strains including Nine Mile strain as antigen. Thus, a combination of PCR and ELISA is the best tool for diagnosis of Q fever and the identification of flocks and animals shedding C. burnetii. This improvement of the sensitivity of diagnostic tests probably contributes to the general opinion that Q fever is an emerging or re-emerging disease.
4. Shedding of C. Burnetii in infected flocks We have used PCR to follow shedding of C. burnetii in milk, vaginal mucus and faeces of females of naturally infected sheep, goats and cattle. Ewes were found to shed more often and for a longer time C. burnetii in vaginal mucus and faeces than cows; however, cows and goats shed more often and for a longer time C. burnetii in milk.
5. Control of the disease Several actions may be proposed to prevent and reduce the animal and environmental contamination. Placentae and foetuses must be destroyed, in order to prevent their ingestion by domestic or wild carnivores, which may disseminate the disease. In addition, manure must be treated with lime or calcium cyanide 0.4% (Arricau-Bouvery et al., 2001) before spreading on fields; this must be done in the absence of wind to avoid propagation of the microorganism faraway. Antibiotic treatments may be performed to reduce the number of abortions and the quantity of C. burnetii shed at parturition. They generally consist in administering two injections of oxytetracycline (20 mg per kg bodyweight) during the last month of gestation, although this treatment does not totally suppress the abortions and the shedding of C. burnetii at lambing (Berri et al., in press). In ruminants, the only way to prevent the disease is vaccination of animals in infected flocks, as well as in uninfected ones close to them, with an efficient vac-
123
cine preventing abortions and shedding of the bacteria. Several vaccines have been developed for this purpose. However, C. burnetii phase I vaccines are difficult and hazardous to obtain, although described as the only efficient vaccines. Indeed, the efficacy of two commercially available vaccines containing the inactivated C. burnetii reference strain Nine Mile, one phase I (COXEVAC-CEVA) and one phase II (CHLAMYVAX-FQ) were assessed in goats (Arricau-Bouvery et al., 2005). In that trial, 2 months before mating, 17 female goats were subcutaneously vaccinated with the phase I vaccine (group C) and 16 female goats with the phase II vaccine (group M). On the 84th day of gestation, these animals, as well as 14 unvaccinated control female goats (group T) were subcutaneously challenged with 104 C. burnetii strain CbC1, isolated from a case of caprine abortion. The phase I vaccine effectively prevented abortions; only 1/17 goat aborted in group C, whilst 13/15 and 9/12 animals aborted in groups M and T, respectively. Mean length of gestation (±S.E.) was normal for group C: 153 ± 3 days, but too short for groups M and T: 134 ± 15 and 141 ± 8 days, respectively. Kidding performance of group C animals, 22/26 (85%) live kids, was similar to that observed in the flock of origin; respective figures were 7/23 (30%) for group T and 9/18 (33%) for group M. Detection of Coxiella was made by trans-PCR in vaginal mucus, faeces and milk; presence of the organism was detected in vaginal mucus, faeces and milk samples of all goats of group M and T. Furthermore, only 7/17 animals had a transient bacterial shedding in vaginal mucus (for 1.5 day in average, compared for 16 and 22 days in group M and T animals, respectively) and 12/17 in faeces (for 10 days in average, compared for 28 and 27 days in group M and T animals, respectively). It thus becomes obvious that under these experimental conditions, only COXEVACCEVA vaccine was efficient, by dramatically reducing abortion and excretion of bacteria in milk, vaginal mucus and faeces, reducing environmental contamination and thus, the risk of transmission to humans. In contrast, the phase II vaccine CHLAMYVAX FQ did not modify the course of the disease. It is thus evident that a phase I vaccine must be used to control the disease and to reduce environmental contamination and thus, the risk of transmission to humans. The widespread application of such a vaccine in cattle in Slovakia in the 1970s and 1980s
124
A. Rodolakis / Small Ruminant Research 62 (2006) 121–124
significantly reduced occurrence of Q fever in that country (Kovacova and Kazar, 2002). Until now, efficacy of this vaccine had not been evaluated in experimental conditions on pregnant ewes; however, in a quantitative mouse model, we have demonstrated that it was as efficient on the CbO1 C. burnetii strain isolated from an aborted ewe, as well as on the CbC1 C. burnetii strain used to challenge pregnant goats.
References Arricau-Bouvery, N., Souriau, A., Bodier, C., Dufour, P., Rousset, E., Rodolakis, A., 2005. Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats. Vaccine 23, 4392–4402. Arricau-Bouvery, N., Souriau, A., Moutoussamy, A., Ladenise, K., Rodolakis, A., 2001. Etude de l’excr´etion de Coxiella burnetii dans un mod`ele exp´erimental caprin et d´econtamination des lisiers par la cyanamide calcique. Renc. Rech. Rumin 8, 153– 156. Berri, M., Souriau, A., Crosby, M., Rodolakis, A., 2002. Shedding of Coxiella burnetii in ewes in two pregnancies following an episode
of Coxiella burnetii abortion in a sheep flock. Vet. Microbiol. 85, 55–60. Berri, M., Arricau-Bouvery, N., Rodolakis, A., 2003. PCR-based detection of Coxiella burnetii from clinical samples. In: Sachse, K., Frey, J. (Eds.), Methods in Molecular Biology. Humana Press Inc., Totowa, NJ, pp. 153–160. Berri, M., Crochet, D., Santiago, S., Rodolakis, A., in press. Spread of Coxiella burnetii infection in sheep flock and their lamb after a Q fever episode. Vet. Rec. Hackstadt, T., 1986. Antigenic variation in the phase I lipopolysaccharides of Coxiella burnetii isolates. Infect. Immun. 52, 337–340. Kovacova, E., Kazar, J., 2002. Q fever—still a query and underestimated infectious disease. Acta Virol. 46, 193–210. Kovacova, E., Kazar, J., Spanelova, D., 1998. Suitability of various Coxiella burnetii antigen preparations for detection of serum antibodies by various tests. Acta Virol. 42, 365–368. Raoult, D., Vestris, G., Enea, M., 1990. Isolation of 16 strains of Coxiella burnetii from patients by using a sensitive centrifugation cell culture system and establishment of the strains in HEL cells. J. Clin. Microbiol. 11, 2482–2484. To, H., Hotta, A., Zhang, G.Q., Nguyen, S.V., Ogawa, M., Yamaguchi, T., Fukushi, H., Amano, K., Hirai, K., 1998. Antigenic characteristics of polypeptides of Coxiella burnetii isolates. Microbiol. Immunol. 42, 81–85.
Q fever, state of art: Epidemiology, diagnosis and prophylaxis夽 A. Rodolakis ∗ INRA, Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France Available online 22 September 2005
Abstract Q fever is a widespread zoonosis caused by Coxiella burnetii. The respiratory tract is the most common route of infection, which occurs by inhalation of contaminated dust and spray shed from infected animals. Livestock is considered as the major “source” for human infections. This short review summarizes the state of the knowledge of the diagnosis, treatment and prevention of Q fever in sheep flocks. © 2005 Elsevier B.V. All rights reserved. Keywords: Sheep; Abortion; Coxiella burnetii; Vaccination; Control
1. Introduction Q fever (for query fever), a zoonosis caused by the obligate intracellular microorganism Coxiella burnetii, is endemic throughout the world and infects arthropods, birds, pets, domestic and wild mammals, as well as humans. The disease is known since the 1930s and has a worldwide distribution, with the exception of the Antarctica and possibly New Zealand. In ewes, C. burnetii infections are generally asymptomatic, but they can lead to abortions, stillbirths and delivery of weak and unviable lambs. In the majority of cases, abortions occur at the end of gestation without specific clinical signs until abortion is imminent. 夽 This paper is part of a special issue entitled Keynote Lectures of
the 6th International Sheep Veterinary Congress—Guest Edited by Dr. George C. Fthenakis and Prof. Quintin A. McKellar. ∗ Tel.: +33 24741 7700; fax: +33 247 427 7779. E-mail address: [email protected]. 0921-4488/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2005.07.038
Such reproductive failures are accompanied with shedding of great number of Coxiella into birth products, urine, faeces and milk of infected animals. Aborted foetuses appear normal, but infected placentas exhibit intercotyledonary fibrous thickening and discolored exudates, which are not specific to Q fever. The abortion rate is generally low. It can range from 3 to 80% of pregnant female, but the higher abortion percentages are rarely observed, except in some goat herds. In sheep flocks, the number of females, which abort, may not be enough to alert the farmer and human clinical cases often reveal the ovine infection. In humans, the acute disease appears like a flu-like infection, usually self-limiting illness accompanied by myalgia and severe headache. Complications, such as pneumonia or hepatitis, may also occur. Endocarditis in patients suffering from valvulopathy, as well as premature delivery or abortion in pregnant women, are the main severe manifestations of the chronic evolution of the disease.
122
A. Rodolakis / Small Ruminant Research 62 (2006) 121–124
The source of human infection is often unidentified, although sheep and goats are more frequently involved in the disease cycle than other animal species. The main route of C. burnetii infection is by inhalation of contaminated aerosols or dusts containing the microorganism shed from infected animals. As C. burnetii is very stable in the environment, resisting to elevated temperature, desiccation, osmotic shock, ultra-violet light and disinfectants, direct contact with an aborting female is not required. People may be infected by handling contaminated wool, manure or clothes contaminated with feces or by the transhumance of infected flocks through a valley. The airborne transmission of C. burnetii associated with its resistance and the ability of producing easily a large quantity of organisms in the placenta of an aborting ewe or doe, have contributed to consider C. burnetii as a category B biological terrorist agent, although the great number of asymptomatic infections limits the consequence of its use as biological weapon. Oral transmission, by ingestion of contaminated raw milk or dairy products in particular goat dairy products could lead to seroconversion and in few cases to Q fever.
2. Antigenic variations of C. burnetii C. burnetii is unique among rickettsiae in that its lipopolysaccharide (LPS) presents a phase variation, similar to smooth-rough variation in the LPS of enterobacteria. Virulent C. burnetii designed phase I corresponds to “smooth-type” LPS and is infectious for animals and humans contrary to phase II C. burnetii obtained after several passages in chicken embryos or cells culture. Phase II C. burnetii is more readily phagocytosed than is phase I C. burnetii, but it is killed by complement contrary to phase I organisms, which resist to complement-mediated serum killing. Phase II C. burnetii has a truncated LPS typical of “rough” LPS: the carbohydrate portion of phase II LPS lacks many of the sugars present in phase I LPS. In addition to phases I–II LPS antigenic variation, other antigenic variations in LPS (Hackstadt, 1986) and in outer membrane proteins (To et al., 1998) of C. burnetii isolates from various sources have been demonstrated. In particular antigenic variations have been observed between C. burnetii strains isolated from ruminants and the reference Nine Mile C. burnetii
strain isolated from a tick in USA; that could explain some lack of sensibility of ELISA kits using Nine Mile C. burnetii strain as antigen.
3. Diagnosis Since there are no specific signs of Q fever, suitable laboratory tests are required for accurate diagnosis. The diagnosis of Q fever remains difficult and epidemiological studies are often based only on serological investigations, which have the potential disadvantage of indicating exposure rather than the detection of the organism. Routine diagnosis of Q fever is usually established by examination of fixed impressions or smears prepared from the placenta stained by the Stamp, Gimenez or Machiavello methods, associated with serological tests. Isolation of C. burnetii is not performed for routine diagnosis in veterinary medicine, because C. burnetti does not grow on standard laboratory bacteriological media and its isolation takes long time and is difficult and hazardous to perform: it requires confined level 3 laboratories (L3). Therefore, isolation of C. burnetii is restricted to specialized laboratories by using the shell-vial cell culture technique (Raoult et al., 1990) or culture on chicken embryos. Recently, polymerase chain reaction (PCR) has radically changed the diagnosis of Q fever in veterinary medicine (Berri et al., 2003). It is the only one, which allows identifying shedders. PCR kits are becoming available and provide a specific, sensitive and rapid tool for the detection of C. burnetii in various clinical samples. The CF test, which is the OIE prescribed serological test, is weakly sensitive and the antigen used in this test frequently fails to detect antibodies in sheep or goats (Kovacova et al., 1998). Moreover, the cutoff value used for diagnosis of chlamydiosis is often used for Q fever, leading to mistakes. The speciesspecific indirect immunofluorescence assay is not often used for diagnosis of Q fever in animals. The ELISA test is more sensitive than the CF test and allows for testing a greater number of animals and flocks. However, this does not allow individual identification of animals that shed C. burnetii in faeces or milk. If most animals, which excrete C. burnetii in vaginal mucus, faeces or milk, are seropositive, some animals can be seropositive without shedding C. burnetii and a few
A. Rodolakis / Small Ruminant Research 62 (2006) 121–124
ones can excrete and remain seronegative (Berri et al., 2002). This last situation has an important consequence for animal and public health, and is due to the antigen used in this test instead of a lack of antibodies response as we have demonstrated it by using ELISA or immunofluorescence assay with various C. burnetii strains including Nine Mile strain as antigen. Thus, a combination of PCR and ELISA is the best tool for diagnosis of Q fever and the identification of flocks and animals shedding C. burnetii. This improvement of the sensitivity of diagnostic tests probably contributes to the general opinion that Q fever is an emerging or re-emerging disease.
4. Shedding of C. Burnetii in infected flocks We have used PCR to follow shedding of C. burnetii in milk, vaginal mucus and faeces of females of naturally infected sheep, goats and cattle. Ewes were found to shed more often and for a longer time C. burnetii in vaginal mucus and faeces than cows; however, cows and goats shed more often and for a longer time C. burnetii in milk.
5. Control of the disease Several actions may be proposed to prevent and reduce the animal and environmental contamination. Placentae and foetuses must be destroyed, in order to prevent their ingestion by domestic or wild carnivores, which may disseminate the disease. In addition, manure must be treated with lime or calcium cyanide 0.4% (Arricau-Bouvery et al., 2001) before spreading on fields; this must be done in the absence of wind to avoid propagation of the microorganism faraway. Antibiotic treatments may be performed to reduce the number of abortions and the quantity of C. burnetii shed at parturition. They generally consist in administering two injections of oxytetracycline (20 mg per kg bodyweight) during the last month of gestation, although this treatment does not totally suppress the abortions and the shedding of C. burnetii at lambing (Berri et al., in press). In ruminants, the only way to prevent the disease is vaccination of animals in infected flocks, as well as in uninfected ones close to them, with an efficient vac-
123
cine preventing abortions and shedding of the bacteria. Several vaccines have been developed for this purpose. However, C. burnetii phase I vaccines are difficult and hazardous to obtain, although described as the only efficient vaccines. Indeed, the efficacy of two commercially available vaccines containing the inactivated C. burnetii reference strain Nine Mile, one phase I (COXEVAC-CEVA) and one phase II (CHLAMYVAX-FQ) were assessed in goats (Arricau-Bouvery et al., 2005). In that trial, 2 months before mating, 17 female goats were subcutaneously vaccinated with the phase I vaccine (group C) and 16 female goats with the phase II vaccine (group M). On the 84th day of gestation, these animals, as well as 14 unvaccinated control female goats (group T) were subcutaneously challenged with 104 C. burnetii strain CbC1, isolated from a case of caprine abortion. The phase I vaccine effectively prevented abortions; only 1/17 goat aborted in group C, whilst 13/15 and 9/12 animals aborted in groups M and T, respectively. Mean length of gestation (±S.E.) was normal for group C: 153 ± 3 days, but too short for groups M and T: 134 ± 15 and 141 ± 8 days, respectively. Kidding performance of group C animals, 22/26 (85%) live kids, was similar to that observed in the flock of origin; respective figures were 7/23 (30%) for group T and 9/18 (33%) for group M. Detection of Coxiella was made by trans-PCR in vaginal mucus, faeces and milk; presence of the organism was detected in vaginal mucus, faeces and milk samples of all goats of group M and T. Furthermore, only 7/17 animals had a transient bacterial shedding in vaginal mucus (for 1.5 day in average, compared for 16 and 22 days in group M and T animals, respectively) and 12/17 in faeces (for 10 days in average, compared for 28 and 27 days in group M and T animals, respectively). It thus becomes obvious that under these experimental conditions, only COXEVACCEVA vaccine was efficient, by dramatically reducing abortion and excretion of bacteria in milk, vaginal mucus and faeces, reducing environmental contamination and thus, the risk of transmission to humans. In contrast, the phase II vaccine CHLAMYVAX FQ did not modify the course of the disease. It is thus evident that a phase I vaccine must be used to control the disease and to reduce environmental contamination and thus, the risk of transmission to humans. The widespread application of such a vaccine in cattle in Slovakia in the 1970s and 1980s
124
A. Rodolakis / Small Ruminant Research 62 (2006) 121–124
significantly reduced occurrence of Q fever in that country (Kovacova and Kazar, 2002). Until now, efficacy of this vaccine had not been evaluated in experimental conditions on pregnant ewes; however, in a quantitative mouse model, we have demonstrated that it was as efficient on the CbO1 C. burnetii strain isolated from an aborted ewe, as well as on the CbC1 C. burnetii strain used to challenge pregnant goats.
References Arricau-Bouvery, N., Souriau, A., Bodier, C., Dufour, P., Rousset, E., Rodolakis, A., 2005. Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats. Vaccine 23, 4392–4402. Arricau-Bouvery, N., Souriau, A., Moutoussamy, A., Ladenise, K., Rodolakis, A., 2001. Etude de l’excr´etion de Coxiella burnetii dans un mod`ele exp´erimental caprin et d´econtamination des lisiers par la cyanamide calcique. Renc. Rech. Rumin 8, 153– 156. Berri, M., Souriau, A., Crosby, M., Rodolakis, A., 2002. Shedding of Coxiella burnetii in ewes in two pregnancies following an episode
of Coxiella burnetii abortion in a sheep flock. Vet. Microbiol. 85, 55–60. Berri, M., Arricau-Bouvery, N., Rodolakis, A., 2003. PCR-based detection of Coxiella burnetii from clinical samples. In: Sachse, K., Frey, J. (Eds.), Methods in Molecular Biology. Humana Press Inc., Totowa, NJ, pp. 153–160. Berri, M., Crochet, D., Santiago, S., Rodolakis, A., in press. Spread of Coxiella burnetii infection in sheep flock and their lamb after a Q fever episode. Vet. Rec. Hackstadt, T., 1986. Antigenic variation in the phase I lipopolysaccharides of Coxiella burnetii isolates. Infect. Immun. 52, 337–340. Kovacova, E., Kazar, J., 2002. Q fever—still a query and underestimated infectious disease. Acta Virol. 46, 193–210. Kovacova, E., Kazar, J., Spanelova, D., 1998. Suitability of various Coxiella burnetii antigen preparations for detection of serum antibodies by various tests. Acta Virol. 42, 365–368. Raoult, D., Vestris, G., Enea, M., 1990. Isolation of 16 strains of Coxiella burnetii from patients by using a sensitive centrifugation cell culture system and establishment of the strains in HEL cells. J. Clin. Microbiol. 11, 2482–2484. To, H., Hotta, A., Zhang, G.Q., Nguyen, S.V., Ogawa, M., Yamaguchi, T., Fukushi, H., Amano, K., Hirai, K., 1998. Antigenic characteristics of polypeptides of Coxiella burnetii isolates. Microbiol. Immunol. 42, 81–85.