Prevalence and risk factors for Coxiella burnetii (Q fever) in Dutch dairy cattle herds based on bulk tank milk testing

Despite cattle herds can harbor Coxiella burnetii, risk factors for C. burnetii presence in dairy cattle herds are largely unknown. Therefore, C. burnetii herd prevalence and risk factors for bulk tank milk (BTM) positivity were investigated. In this cross-sectional study, a questionnaire was filled out by the farmer and BTM from 301 farms was tested by ELISA for presence of C. burnetii antibodies and PCR for presence of C. burnetii DNA. Risk factors were identified by univariable and multivariable logistic regression analyses. Antibodies to C. burnetii were detected in 81.6% (CI: 77.2–85.9) and C. burnetii DNA in 18.8% (CI: 14.4–23.1) of the BTM samples. Herd size (OR = 1.1 per 10 cows), cleaning the bedding of the cubicles at most every other day (OR = 2.8) and purchase of cattle from at least two addresses (OR = 3.1) showed a significant and positive association with ELISA positivity and use of an automatic milking system a negative association (OR = 0.3). Risk factors for PCR positivity were purchase of cattle from at least two delivery addresses (OR = 3.2), presence of cows with ticks (OR = 2.0), use of an automatic milking system (OR = 0.2) and presence of goats or sheep on the farm (OR = 0.4). Biosecurity and general hygiene seem associated with introduction and spread of C. burnetii in dairy herds.     

Occurrence of Coxiella burnetii in Polish dairy cattle herds based on serological and PCR tests

The aim of the research was to assess the prevalence of antibodies to Coxiella burnetii in dairy cattle herds in Poland and to compare the results of real-time PCR and ELISA tests performed on bulk tank milk (BTM) samples. In total, 2635 serum samples collected from 969 dairy cattle herds from all provinces were tested using ELISA. Additionally, BTM specimens from 101 herds were analysed by ELISA and real-time PCR targeting IS1111 element. Presence of anti-C. burnetii antibodies was confirmed in 25.39% of serum samples in 237 herds (24.46%) and the herd-level seroprevalence in Voivodeships varied from 2.5% to 61.4%. Moreover, 46 (45.5%) of analysed bulk tank milk samples gave postive result in ELISA and microbial DNA was detected in 40 (39.6%) of tested herds. The comparative analysis of ELISA and real-time PCR results obtained for BTM samples using the chi-square test showed statistically significant relationship between results of both methods.     

Surveys on Coxiella burnetii infections in Swedish cattle,sheep, goats and moose

Background

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Prevalence data in ruminant species are important to support risk assessments regarding public and animal health. The aim was to investigate the presence of or exposure to C. burnetii in cattle, sheep, goats and moose, and to compare two enzyme-linked immunosorbent assays (ELISAs). National surveys of antibodies against C. burnetii were performed for dairy cattle (n=1537), dairy goats (n=58) and sheep (n=518). Bovine samples consisted of bulk milk, caprine of pooled milk, and ovine of pooled serum. Antibodies were investigated in moose samples (n=99) from three regions. A one-year regional cattle bulk milk survey was performed on the Isle of Gotland (n=119, four occasions). Cattle, sheep and goat samples were analysed with indirect ELISA and moose samples with complement fixation test. For the sheep, goat, and parts of the cattle survey, samples were run in parallel by ELISAs based on antigens from infected ruminants and ticks. Bulk milk samples from the regional cattle survey and vaginal swabs from a subset of the sheep herds (n=80) were analysed for the agent by polymerase chain reaction. Spatial clustering was investigated in the national cattle survey.

Results

The prevalence of antibodies in dairy herds was 8.2% with large regional differences. High risk clusters were identified in the southern regions. The prevalence among dairy herds on the Isle of Gotland varied from 55.9% to 64.6% and 46.4% to 58.9.0% for antibodies and agent, respectively, overall agreement between agent and antibodies was 85.2%. The prevalence of antibodies in sheep was 0.6%, the agent was not detected the vaginal swabs. Antibodies were not detected in goats or moose, although parts of the moose samples were collected in an area with high prevalence in cattle. The overall agreement between the two ELISAs was 90.4%.

Conclusions

The prevalence of antibodies against C. burnetii in dairy cattle in Sweden shows large regional differences. The results suggest that C. burnetii is a rare pathogen among Swedish moose, dairy goat and sheep. ELISAs based on ruminant and tick antigen performed in a similar manner under Swedish conditions.     

Increasing prevalence of Coxiella burnetii seropositive Danish dairy cattle herds

A study based on bulk tank milk samples from 120 randomly selected dairy cattle herds was conducted to estimate the prevalence of Coxiella burnetii seropositive dairy herds, to describe the geographical distribution, and to identify risk factors. Using the CHEKIT Q-fever Antibody ELISA Test Kit (IDEXX), the study revealed a prevalence of 79.2% seropositive herds, 18.3% seronegative herds, and 2.5% serointermediate herds based on the instructions provided by the manufacturer. Multifactorial logistic regression showed statistically significant associations (P < 0.01) between C. burnetii seropositivity and increasing herd size (OR = 1.02 per cow increment) and increasing regional average number of cattle per dairy herd (OR = 1.02 per animal increment). Herds >150 cows had 17.9 times higher odds of testing positive compared to herds <80 cows. The regional average number of cattle herds per square kilometer was borderline significantly related to the occurrence of seropositive dairy herds (P = 0.06). The results indicate an increased prevalence of seropositive dairy herds since the previous survey in 2008 and an adverse impact of increasing herd size and cattle density on the risk of seropositivity.     

Re-emergence of trichinellosis in southeastern Europe due to political and economic changes

The countries of southeastern Europe including the Balkan region and bordering countries - Albania, Bulgaria, Bosnia and Herzegovina, Croatia, Greece, Hungary, Macedonia, Romania, Serbia and Montenegro, Slovenia, and the European part of Turkey - occupy a very important strategic position and represent a land bridge between Europe and Asia. In the majority of southeastern European countries, cases of trichinellosis among the human and animal populations were described in the late 19th or early 20th centuries. Trichinella infections among wildlife were also described in the aforementioned countries. Today, the prevalence of trichinellosis is different between the Balkans and bordering countries. A high prevalence of trichinellosis in domestic animals and humans has been reported in Bulgaria, Serbia and Montenegro, Romania and Croatia. A moderate prevalence was found in Bosnia and Herzegovina. In Hungary, human trichinellosis has not been present for a long period of time. However, sporadic cases were recorded in swine over the last 2 years. Trichinellosis has not been found among domestic animals and humans in Greece and Macedonia in recent years while in Turkey and Slovenia human trichinellosis is sporadic. The re-emergence of trichinellosis is connected with the changes in the social and political systems in Bulgaria and Romania. In Serbia and Montenegro as well in Croatia, however, a re-emergence of trichinellosis was due not only to political and social changes but also to wars that took place in these countries during the last years of the 20th century. Social, economic and political factors responsible for the re-emergence of trichinellosis in southeast European countries are discussed in this communication.     

Herd prevalence and genotypes of Coxiella burnetii in dairy cattle bulk tank milk in Gyeongsang provinces of South Korea

Coxiella burnetii is the causative agent of the zoonotic Q fever, and its reservoirs include ticks and livestock, which are key sources of transmission to humans. Although there have been several studies on the prevalence of C. burnetii antibodies in dairy cattle bulk tank milk (BTM), there is a lack of information on the molecular detection of C. burnetii in BTM in South Korea. Thus, this study was designed to assess milk shedding of C. burnetii in BTM from dairy cattle herds. Among the 607 BTM samples collected from 41 counties in Gyeongsang provinces in 2015, 108 (17.8%) from 23 (56.1%) counties tested positive for C. burnetii by PCR. Because the 16S rRNA sequences of C. burnetii from all 108 PCR-positive samples were identical, two representative samples (BTM-GB-10 and BTM-GN-63) are described in this paper. These sequences showed high identity (96.9–100%) to other C. burnetii sequences deposited in GenBank. Phylogenetic analysis showed that these two sequences were clustered with existing C. burnetii strains. The relatively high prevalence rates of C. burnetii in BTM detected in this study suggest that C. burnetii is prevalent among dairy cattle herds in South Korea. Thus, implementation of continuous monitoring and control strategies for domestic animals is needed to prevent disease transmission and protect public health.

     

Prevalence of Coxiella burnetii antibodies in Portuguese dairy cattle herds

Q fever is an important zoonotic disease which has been recently diagnosed, mainly in sheep and goats, in Portugal. The aim of the present study was to determine the prevalence of bovine Coxiella burnetii antibodies in dairy farms from the northwest of Portugal. Bulk tank milk samples were randomly obtained, on November 2013, from 90 dairy farms and assayed using an ELISA kit. The apparent prevalence was 61.1 % (95 % C.I. from 50.8 to 70.5 %). The proportion of negative and intermediate (inconclusive) herds was 34.5 % (25.5 to 44.7 %) and 4.4 % (1.7 to 10.9 %), respectively. In conclusion, a high level of exposure to Coxiella burnetii was observed in Portuguese dairy cattle herds, highlighting the needs to better understand the epidemiology of Q fever in Portugal by the implementation of a monitoring program based on harmonized serologic and molecular methodologies and elucidation of the infection status of the herds.

     

The first serologic study of Q fever in sheep in Iran

Coxiella burnetii is an obligate intracellular microorganism that causes Q fever in humans and animals. In ewes, C. burnetii infections are generally asymptomatic, but they can lead to abortions, stillbirths, and delivery of weak and unviable lambs. Serological assays are suitable for screening herds. Enzyme-linked immunosorbent assays (ELISA) technique has a high sensitivity and a good specificity. The aim of this study was to investigate the presence of anti-C. burnetii antibodies among sheep in southeast Iran. A total of 85 serum samples were collected from ten sheep flocks from April to September 2009. Serum samples were tested for Q fever antibodies using a commercial indirect ELISA kit. Antibodies were detected in 25 sera (29.42%) of 85 samples. Sixteen female (18.82%) and nine male (10.58%) cases had antibodies specific to C. burnetii. There is significant difference in seropositivity between male and female groups (P < 0.05). This first study of C. burnetii seroprevalence in sheep in southeast Iran has indicated that seropositive animals can be found throughout the country. Further work is now required to characterize the epidemiology of the infection more thoroughly.     

Serologic and molecular survey of horses to Coxiella burnetii in East of Iran a highly endemic area

There are few reports about Q fever in horse populations worldwide. This study aimed to detect the C. burnetii infection by serologic and molecular confirmation using commercial ELISA kit and real-time PCR in the East of Iran a region highly endemic. A total of 177 blood samples and 115 vaginal swabs were randomly collected from horses in East of Iran. The sera samples were analyzed for anti C.burnetii Ig G antibodies by a commercial ELISA kit and nucleic acid extraxted from vaginal samples were used to determine the C. burnetii DNA by real-time PCR assay. Antibodies were detected in 5.64 % (10/177) of sera samples and C. burnetii DNA was detected in 7.82 % (9/115) of horse vaginal samples. There was no significant difference in seroprevalence in different sex, age and breed groups. Our study showed that horses could be considered as a mild potential reservoir of C. burnetii which may be effective on horse health status. However, additional studies are needed to assess whether the horse could be considered as a relevant transmission risk indicator for Q fever.     

Risk factors for Coxiella burnetii antibodies in bulk tank milk from Danish dairy herds

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR = 3.6), human contacts (OR = 4.2), artificial insemination by other people than artificial insemination technicians (OR = 7.7), routine herd health contract with the veterinarian (OR = 4.3) and hygiene precautions taken by veterinarians (OR = 5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii.     

Occurrence and risk factors of Coxiella burnetii in domestic ruminants in Lebanon

Coxiella burnetii causes diseases in humans (Q fever) and animals, domestic ruminants playing a major role in the epidemiology of the infection. Information on C. burnetii infection in Lebanon is scanty. In order to assess the prevalence of C. burnetii infection in ruminants, a cross-sectional study was undertaken in 2014. A total of 1633 sera from ruminants (865 cattle, 384 sheep and 384 goats) from 429 farms (173 cattle, 128 sheep and 128 goats), in seven provinces of Lebanon were randomly selected and assayed for the presence of antibodies.39.86% of farms (95% CI: 35.23–44.56) resulted positive. The seroprevalence was 30.63% in Cattle-farms, 46.88% in sheep-farms and 45.31% in goat-farms.Milk samples collected from 282 seropositive animals (86 cows, 93 sheep and 103 goats) from 171 positive farms were tested by a high sensitive Real-Time PCR targeted to the IS1111 transposon of C. burnetii. The overall prevalence in farms was estimated to be 14.04%. Cattle-, sheep- and goat farm prevalence rates were 15.09%, 10% and 17.24%, respectively.The findings of the study show that C. burnetii prevalence in Lebanese domestic ruminants is related to animal species and farming practices. Indeed, the mixed herds with sheep (p < 0.01), the presence of common lambing/kidding areas (p < 0.001) in farms where the use of disinfectants was not a routine practice (p < 0.05) were identified as important risk factors.The results of the study provide baseline information for setting up herd management and public health measures for the prevention and control of Q fever in Lebanon.     

High prevalence and risk factors of Coxiella burnetii in milk of dairy animals with a history of abortion in Iran

Coxiella burnetii is causative agent of Q fever, which is a public health problem in most countries. The aim of this study was to study the prevalence rate of C. burnetii in raw milk of dairy animals in Iran with previous history of abortion. In this survey, milk samples were collected from different dairy animals with history of abortion from Qom province (center of Iran). Samples were tested by Nested PCR and Real-time PCR for detection of IS1111 gene of C. burnetii. In total, 34.92% (44 of 126) milk samples were positive for C. burnetii. Prevalence of C. burnetii in cattle, sheep and goat milk was 33.33%, 35.71% and 35.71%, respectively. Age was a significant risk factor for shedding of C. burnetii in cattle (P = 0.02) and goat (P = 0.05). Shedding of C. burnetii was high prevalence in milk of dairy animals with history of abortion in Iran. The high prevalence of this bacterium in milk (especially in animals with history of abortion) indicates that Excreted by milk as a potential source to spread of infection in the environment.     

Serosurvey of Coxiella burnetii (Q fever) in Dromedary Camels (Camelus dromedarius) in Laikipia County,Kenya

Dromedary camels (Camelus dromedarius) are an important protein source for people in semi‐arid and arid regions of Africa. In Kenya, camel populations have grown dramatically in the past few decades resulting in the potential for increased disease transmission between humans and camels. An estimated four million Kenyans drink unpasteurized camel milk, which poses a disease risk. We evaluated the seroprevalence of a significant zoonotic pathogen, Coxiella burnetii (Q fever), among 334 camels from nine herds in Laikipia County, Kenya. Serum testing revealed 18.6% positive seroprevalence of Coxiella burnetii (n = 344). Increasing camel age was positively associated with C. burnetii seroprevalence (OR = 5.36). Our study confirmed that camels living in Laikipia County, Kenya, have been exposed to the zoonotic pathogen, C. burnetii. Further research to evaluate the role of camels in disease transmission to other livestock, wildlife and humans in Kenya should be conducted.     

Seroprevalence and Factors Associated with Coxiella burnetii Infection in Small Ruminants in Baringo County,Kenya

To improve estimates of C. burnetii epidemiology in Kenya, a survey was undertaken in small ruminants in Baringo County, where acute cases of Q fever in humans had been reported in 2014. From 140 household herds selected, 508 (60.5%) goats and 332 (39.5%) sheep were included and an indirect ELISA assay for C. burnetii IgG antibodies performed. In addition, epidemiological information at both herd and animal level was collected. Generalized mixed‐effects multivariable logistic model using herd as the random effect was used to determine variables correlated to the outcome. Overall seroprevalence was 20.5% (95% CI: 17.8%, 23.3%). Goats had 26.0% (95% CI: 22.2%, 30.0%) compared to sheep 12.2% (95% CI: 8.7%, 16.0%). Nomadic pastoralism, goats and older animals (>1 year) were associated with greater risk of C. burnetii seropositivity (P = ≤0.05). Heterogeneity in C. burnetii seropositivity was observed across the sublocations (P = 0.028). Evidence of C. burnetii exposure in small ruminants revealed poses a potential risk of exposure to the people living in close proximity to the animals. We recommended integrated animal–human surveillance and socio‐economic studies for C. burnetii, to aid our understanding of the risk of transmission between the animals and humans, and in the design of prevention and control strategies for the disease in the region.     

Seroprevalence and risk factors for C. burentii infection in camels in Egypt

Q fever caused by Coxiella burentii, gram negative obligate intracellular bacterium. The disease has been reported in wide range of animals especially ruminants. The available data about the prevalence of Q fever in camels in Egypt are limited. Therefore, the present study aimed to investigate the seroprevalence of C. burnetii among camels and identify the risk factors associated with infection. A total 315 serum samples were collected from three governorates in Egypt during 2018 and examined by an indirect Enzyme linked Immunosorbent Assay (ELISA). The obtained results were subsequent analyzed by chi-square and logistic regression. Generally, the seroprevalence of C. burnetii among camels was 22 %. The results revealed that the seroprevalence of C. burnetii increased in aged female camels in comparison with young one and was higher also in female with history of abortion (OR = 4.6, 95%CI: 2.46–8.76). The infection was significantly increased during autumn season (OR = 9.3, 95%CI: 4.23–20.5). Besides, camels in contact with small ruminants showed high level of infection (OR = 1.12, 95%CI: 0.65–1.93) or camel with heavy tick infestation (OR = 1.08, 95%CI: 0.60–1.92). Our report confirms that the seroprevalence of C. burnetii among camels in Egypt and appropriate control measures should be taken to reduce the transmission of infection to other animal species or human.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.     

Seroepidemiological study on Coxiella burnetii and associated risk factors in ruminants at Kurdistan Province,west of Iran

Q fever is zoonotic disease caused by Coxiella burnetii. Ruminants are the main reservoir of this pathogen, which is often asymptomatic but lead to abortion. This study aims to survey the seroprevalence and risk factors of this zoonose among ruminants in Kurdistan province, the west of Iran. 480 blood samples were collected from ruminants including sheep, goats and cows, each 160 samples, in the age groups of <1, ≥1−3, >3−5 year with and without the history of abortion in two groups border and non-border cities in Kurdistan province. Serums were tested by use of indirect ELISA to determine specific antibodies against C. burnetii. The results indicate the seroprevalence of 46.6 % for Q fever. Seroprevalence in sheep, goats and cows were 28.58 % (n = 64), 45.53 % (n = 102) and 25.89 % (n = 58), respectively. Seroprevalence is significantly higher in animals with abortion than in those without such history (P < 0.05). The seroprevalence in the border cities has been significantly higher than other geographical areas (P < 0.05). Seroprevalence had no significant correlation with animal age (P> 0.05). This study is the first seroepidemiological study done on Q fever in ruminants of Kurdistan province, Iran. The results indicate the high seroprevalence of Coxiella burnetii in the area under the study. Therefore, doing an epidemiologically study aimed at isolating C. brunetii in the human population of Kurdistan province is recommended, so that the epidemiological aspect of this pathogen in the people of Kurdistan province be clarified and subsequently disease control and prevention programs be applied.     

Progression of Coxiella burnetii infection after implementing a two-year vaccination program in a naturally infected dairy cattle herd

Background

The high prevalence of Coxiella burnetii infection in dairy cattle herds recently reported and the long survival time of the bacterium in the environment pose a risk to human and animal health that calls for the implementation of control measures at herd level. This study presents the results of a 2-year vaccination program with an inactivated phase I vaccine in a Spanish dairy herd naturally infected with C. burnetii. Calves older than 3 months and non-pregnant heifers and cows were vaccinated in April 2011 and the farm was subsequently visited at a monthly basis for vaccination of recently calved cows and calves that reached the age of 3 months. Annual booster doses were given to previous vaccinated animals as well. The effectiveness of the vaccine was assessed in terms of level of C. burnetii shedding through milk and uterine fluids and environmental contamination as determined by polymerase chain reaction (PCR).

Results

The percentage of shedder animals through uterine fluids and milk progressively decreased, and C. burnetii DNA load in bulk-tank milk samples was low at the end of the study. The average seroconversion rate in not yet vaccinated animals, which acted as control group, was 8.6% during the first year and 0% in the second year. DNA of C. burnetii was found in aerosols and dust samples taken in the calving area only at the beginning of the study, whereas slurry samples remained C. burnetii PCR positive for at least 18 months. Multiple Locus Variable number tandem-repeat Analysis identified the same genotype in all C. burnetii DNA positive samples.

Conclusions

In the absence of any changes in biosecurity, the overall reduction of C. burnetii infection in animals to 1.2% milk shedders and the reduced environment contamination found at the end of the study was ascribed to the effects of vaccination together with the culling of milk shedders. Vaccination has to be planned as a medium-long term strategy to suppress risks of re-infection.     

Coxiella burnetii associated reproductive disorders in domestic animals-a critical review

The bacterium Coxiella burnetii has been detected in the fetal membranes, birth fluids and vaginal mucus, as well as in the milk and other excretions of several domestic mammals. The finding of C. burnetii in association with abortion, parturition and in the postpartum period has led to the hypothesis that C. burnetii causes a range of reproductive diseases. This review critically evaluates the scientific basis for this hypothesis in domestic mammals.The review demonstrates a solid evidence for the association between C. burnetii infection and sporadic cases of abortion, premature delivery, stillbirth and weak offspring in cattle, sheep and goats. C. burnetii induced in-herd epidemics of this complete expression of reproductive failure have been reported for sheep and goats, but not for cattle. The single entities occur only as part of the complex and not as single events such as generally increased stillbirth rate. Studies show that C. burnetii initially infects the placenta and that subsequent spread to the fetus may occur either haematogenous or by the amniotic-oral route. The consequences for the equine, porcine, canine and feline conceptus remains to the elucidated but that infection of the conceptus may occur is documented for most species. There is no solid evidence to support a hypothesis of C. burnetii causing disorders such as subfertility, endometritis/metritis, or retained fetal membranes in any kind of domestic animal species.There is a strong need to validate non-pathology based methods such as polymerase chain reaction for their use in diagnostic and research in relation to establishing C. burnetii as the cause of abortion and to adapt an appropriate study design and include adequate control animals when linking epidemiological findings to C. burnetii or when evaluating effects of vaccination in production herds.     

Detection of Coxiella burnetii, the agent of Q fever, in oviducts and uterine flushing media and in genital tract tissues of the non pregnant goat

The aim of the present study was the detection and quantification of Coxiella burnetii DNA in the flushing media (oviducts and uterine horns) and genital tract tissues of non pregnant goats from 20 goats chosen at random from 86 goats originating from 56 different breeding herds in south-west France. The serological prevalence rate of C. burnetii in the study population was 70.3%.The DNA of C. burnetii was identified using conventional PCR in the flushing media from the oviducts and uterus in 8/20 goats (40%) and in genital tract tissues (oviduct, uterus and ovary) in 5/20 goats (25%). This study clearly shows for the first time that the media used to flush the oviducts or uterine horns, collected using the standard embryo harvesting technique in goats, are susceptible to infection with C. burnetii. The 16 conventional PCR-positive samples were also analyzed using real-time PCR. The bacterial load of the oviduct and uterine flushing media varied from 2.9 × 10⁴ to 7.5 × 10⁶ bacteria per flushing medium, while the bacterial load of the tissue samples varied from 1.0 × 10² to 1.5 × 10⁵ bacteria per mg of tissue. The infection of genital tract flushing media and tissues is a risk factor for the transmission of C. burnetii from donor to recipient during embryo transfer or to the embryo and fetus when gestation is pursued to term.