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J Clin Microbiol. 2012 Jun; 50(6): 2156–2158.
PMCID: PMC3372143

Genotyping Reveals the Presence of a Predominant Genotype of Coxiella burnetii in Consumer Milk Products

Abstract

Real-time PCR shows the widespread presence of Coxiella burnetii DNA in a broad range of commercially available milk and milk products. MLVA genotyping shows that this is the result of the presence of a predominant C. burnetii genotype in the dairy cattle population.

TEXT

Q fever is a zoonosis caused by the pathogen Coxiella burnetii, which is prevalent throughout the world (1). Ruminants (sheep, goats, and cattle) are often asymptomatic carriers of C. burnetii and are considered to be a source of infection to humans (1). C. burnetii can cause abortion in small ruminants such as sheep and goats and may cause reproductive disorders in cattle (7). Huge numbers of C. burnetii can be released into the environment via birth products (6). Lower numbers are usually shed in milk, even in asymptomatic herds (2, 3, 4, 5, 8, 9). Although consumption of raw or insufficiently pasteurized milk is very rarely identified as a source of Q fever infection, asymptomatic cattle herds can be considered potential C. burnetii reservoirs capable of transmitting the disease to humans.

We applied real-time PCR, targeting the multicopy IS1111a insertion element of C. burnetii as described earlier (11), and a 6-locus multiple-locus variable number tandem repeat analysis (MLVA) panel (12) to a broad range of milk and milk products with the aim to determine the prevalence and genotypes of C. burnetii in milk (Table 1). The study included commercially available semi-skimmed milk samples from cows (obtained from large supermarket chains) and milk products, such as coffee creamer, obtained throughout Europe and from an additional 10 non-European countries. Samples were collected from different brands, and according to the information on the packages they were produced by the (local) dairy industry in these countries. The origin of the milk samples from Egypt, Saudi-Arabia, and Qatar could not be identified.

Table 1
Prevalence of C. burnetii DNA in commercially available bulk tank cow milk and milk products from 18 countries throughout Europe and from 10 non-European countriesa

Eighty-eight out of 116 (76%) milk samples or milk products from 28 countries contain significant amounts of C. burnetii DNA (Table 1). No C. burnetii DNA was detected in milk obtained from Finland, Norway, Costa Rica, and New Zealand. MLVA genotypes I to O were identified in samples from France, Germany, The Netherlands, Portugal, Slovak Republic, Spain, Switzerland, United Kingdom, Qatar, and Saudi Arabia. MLVA genotypes P, Q, and R were identified in samples from Slovak Republic, Qatar, and Russia, respectively. A partial MLVA genotype (Table 1, “Part”) was obtained from samples that contained insufficient DNA to obtain a full profile. In 4 samples from Slovak Republic, we observed more than one allele per locus, suggesting the presence of at least two or more different genotypes in these samples (Table 1). Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between the MLVA genotypes I to O (Fig. 1). These MLVA genotypes are interconnected by repeat number changes in only one of the six markers and may represent microvariants of one founder genotype. In contrast, MLVA genotypes P and R and the genotypes of five sequenced C. burnetii strains all differed in at least 3 markers from the MLVA genotypes I to O.

Fig 1
Minimum spanning tree showing the relationship between the obtained MLVA genotypes identified in this study and five sequenced C. burnetii strains, i.e., Dugway (GenBank accession number ...

The MLVA genotypes were compared to an in-house database containing 57 different C. burnetii MLVA genotypes from 197 human, caprine, ovine, and cattle clinical samples obtained from Canada, France, Germany, The Netherlands, Portugal, Spain, and the United States. MLVA genotypes I and J have also been recognized incidentally in 8 human clinical samples (placenta and heart valve) from France and in 2 animal samples (cattle and goats) from The Netherlands. However, very different MLVA genotypes (A to H) were identified in human, ovine, and caprine clinical samples from the Q fever outbreak in The Netherlands using a 6-locus and 10-locus MLVA panel (10, 12), indicating that the Dutch Q fever outbreak is not related to the presence of C. burnetii in cattle.

The presence of highly similar C. burnetii genotypes in consumer milk products may indicate a widespread dissemination of a specific cattle-adapted strain. Alternatively, this genotype may have been introduced into different countries by transport of asymptomatic C. burnetii-positive cattle, as well as by export of milk and milk products from a restricted number of countries to other countries (e.g., Egypt, Saudi Arabia, Qatar) by the dairy industry. By testing bulk milk products instead of milk from individual animals, any positive milk specimen is likely to be diluted with negative milk specimens, leading to an average lower DNA concentration, resulting in higher threshold cycle (CT) values as well as partial genotypes.

This is the first report of genotypic diversity among C. burnetii from cow milk throughout Europe and beyond. Integration of such data in international databases can be instrumental to understand the global epidemiology of Q fever in animals.

In conclusion, this study demonstrated the presence of C. burnetii DNA in a broad range of commercially available cow milk and milk products, indicating a high prevalence of C. burnetii among the dairy cattle population worldwide and a possible clonal spread of C. burnetii among the European dairy cattle population. In addition, since this dominant genotype is only incidentally found in humans, the risk of obtaining Q fever via exposure to infected cattle may be much lower than via exposure to infected small ruminants. The incidental observation of mixed alleles does not exclude the possibility of the presence of other minority genotypes in cattle that may be relevant to humans after all.

ACKNOWLEDGMENTS

We are grateful to K. Grif from the Department for Hygiene, Microbiology, and Social Medicine, Medical University Innsbruck; Austria, R. Toman from the Laboratory for Diagnosis and Prevention of Rickettsial and Chlamydial Infections Institute of Virology, Slovakia; G. Dingemans; drivers of transport company Nabuurs, Haps, The Netherlands; A. S. Santos, Centre for Vectors and Infectious Diseases Francisco Cambournac, National Institutes of Health Douter Ricardo Jorge (CEVDI/INSA), Águas de Moura, Portugal; and all employees from the Department of Medical Microbiology and Infectious Diseases of the Canisius Wilhelmina Hospital Nijmegen, The Netherlands, who contributed to collecting milk and milk products worldwide.

Footnotes

Published ahead of print 11 April 2012

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