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Emerg Infect Dis. May 2012; 18(5): 887–889.
PMCID: PMC3358082

Epidemic Genotype of Coxiella burnetii among Goats, Sheep, and Humans in the Netherlands

To the Editor: The 2007–2010 Q fever epidemic among humans in the Netherlands was among the largest reported in magnitude and duration (1). The increase in human Q fever cases coincided with an increase in spontaneous abortions among dairy goats in the southeastern part of the Netherlands, an area that is densely populated with goat farms (1). Genotypic analyses of the involved isolates could confirm the possible link between the human and animal Q fever cases.

In previous studies, genotypic investigations of human and animal samples in the Netherlands were performed by using a 3-locus multilocus variable-number tandem repeats analysis (MLVA) panel and single-nucleotide polymorphism genotyping, respectively (2,3). The first study, performed on relatively few samples from a minor part of the affected area, showed that farm animals and humans in the Netherlands were infected by different but apparently closely related genotypes. More recently, genotyping by using a 10-locus MLVA panel provided additional information about the genotypic diversity of Coxiella burnetii among ruminants in the Netherlands: 1 dominant MLVA genotype was identified among goats and sheep throughout the entire affected Q fever area (4). A different panel of MLVA markers was applied to human samples (5). Four markers that are shared by both panels showed identical alleles in human and animal samples, again implicating goats and sheep as possible sources of the outbreak.

MLVA, which is based on relatively unstable repetitive DNA elements, is sometimes criticized for producing results that are too discriminatory or difficult to reproduce in different settings (6). Because of their instability, use of tandem repeats as genotyping targets can lead to problems with data interpretation and to overestimation of genotypic diversity by showing small variations in MLVA genotypes in isolates of otherwise identical background.

We used a more stable, sequence-based typing method, multispacer sequence typing (MST), on samples from humans and a group of ruminant animals (goats, sheep, and cattle) to establish a firmer correlation between Q fever cases in humans and animals (7). We identified MST genotypes using a Web-based MST database (http://ifr48.timone.univ-mrs.fr/MST_Coxiella/mst) containing genotypes from several countries in Europe. Ultimately, this study could answer the question of whether the current outbreak situation could have been caused by a specific C. burnetii strain in the ruminant population in the Netherlands.

Real-time PCR-positive specimens from 10 humans and 9 Q fever–positive specimens from goats and sheep collected from various locations throughout the affected area were used (8). We also included Q fever-positive specimens from cattle to rule out cattle as a possible source of Q fever infection. Five samples of cow’s milk and 1 bovine vaginal swab sample were analyzed (Table A1). MST33 was identified in 9 of 10 tested human samples and in the remaining 8 of 9 clinical samples from goats and sheep (Table A1). MST33 has been isolated incidentally in nonoutbreak situations in human clinical samples obtained in France during 1996, 1998, and 1999 and from a placenta of an asymptomatic ewe in Germany during 1992. All samples from cattle in the Netherlands, 1 goat, and cow’s milk contained genotype MST20. Genotype MST20 has also been identified in human clinical samples from France, in a cow’s placenta from Germany isolated in 1992 and in rodents from the United States isolated in 1958. In 1 human bronchoalveolar lavage sample, a novel (partial) MST genotype was found. This may be an incidental Q fever case unrelated to the outbreak situation. Because no historical genotyping data for the period before the outbreak of Q fever in the Netherlands are available, this explanation needs further research.

MST genotyping shows the presence of genotype MST33 in clinical samples from humans, goats and sheep. These results confirm that goats and sheep are the source of human Q fever in the Netherlands. Few worldwide genotyping studies have been conducted, and therefore information about a possible global persistence of this genotype is lacking. This study also indicates that the outbreak among humans is not linked to C. burnetii in cattle, although the infection is widespread among dairy herds in the Netherlands (10), exemplifying that most outbreaks are related to goats and sheep rather than to cattle. In conclusion, the increase in the number of Q fever cases in the Netherlands among humans most likely results from MST33 in the goat population in the Netherlands and could have been facilitated by intensive goat farming in the affected area and its proximity to the human population.

Table A1

Coxiella burnetii MST genotypes from humans and ruminants sampled during the Q fever outbreak, the Netherlands, 2008–2010*
Sample no.HostSourceLocationYearCt valueMST genotype†Cox2Cox5Cox18Cox20Cox22Cox37Cox51Cox56Cox57‡Cox61
Q001SheepVaginal swab1200825.733751–§599432
Q002SheepVaginal swab1200816.3337516599432
Q003SheepVaginal swab1200818.8337516599432
Q004LambThroat swab1200827.9337516599432
Q005LambThroat swab1200829.933751599432
Q006LambThroat swab1200828.933751599432
Q050HumanBAL2200922.433751599432
Q052HumanSputum3200920.733751599432
Q054HumanSputum3200919.433751599432
Q057HumanSputum3200920.633751599432
Q063HumanSputum4200929.63375159942
Q066HumanSputum5200927.73375159942
Q076HumanAorta valve6200917.033751599432
Q084HumanAorta valve7200817.033751599432
Q107HumanAorta valve520109.0337516599432
Q085GoatPlacenta8200918.033751599432
Q087GoatPlacenta9200918.133751599432
Q086GoatPlacenta9200918.0203265441065
Q097CattleSwab10200919.0203265441065
Q090CattleMilk11201032.020326544105
Q091CattleMilk12201032.620326544105
Q093CattleMilk13201031.720326544105
Q096CattleMilk14201033.420326544105
Q123CattleMilk15201031.620326544105
Q056HumanBAL16201028.2New3329
DugwayNACP000733#NANANA2032615441065
RSA331NACP000890#NANANA183816347963
RSA493NAAE016828#NANANA163853416765
CbuG Q212NACP001019#NANANA2121462311111
CbuK Q154NACP001020#NANANA85425153344

*MST, multispacer sequence typing; Ct, cycle threshold; BAL, bronchoalveolar lavage; NA, not applicable.
†MST genotypes were identified by using the MST database (http://ifr48.timone.univ-mrs.fr/MST_Coxiella/mst).
‡Result obtained by using improved amplification primers for Cox57 (9).
§ –, no result was obtained. The lack of results may be explained by the significantly larger PCR product that is targeted, low quantity of DNA or to overall poor performance of the PCR amplification.
¶This combination of 4 alleles has not been observed and justifies the assignment of a new MST genotype.
#GenBank accession number.

Footnotes

Suggested citation for this article: Tilburg JJHC, Roest HIJ, Buffet S, Nabuurs-Franssen MH, Horrevorts AM, Raoult D, et al. Epidemic genotype of Coxiella burnetii among goats, sheep, and humans in the Netherlands [letter]. Emerg Infect Dis [serial on the Internet]. 2012 May [date cited]. http://dx.doi.org.10.3201/eid1805.111907

References

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2. Huijsmans CJJ, Schellekens JJA, Wever PC, Toman R, Savelkoul PHM, Janse I, et al. Single-nucleotide-polymorphism-genotyping of Coxiella burnetii during a Q fever outbreak in the Netherlands. Appl Environ Microbiol. 2011;77:2051–7 10.1128/AEM.02293-10 [PMC free article] [PubMed] [Cross Ref]
3. Klaassen CHW, Nabuurs-Franssen MH, Tilburg JJHC, Hamans MAWM, Horrevorts AM. Multigenotype Q fever outbreaks, the Netherlands. Emerg Infect Dis. 2009;15:613–4 10.3201/eid1504.081612 [PMC free article] [PubMed] [Cross Ref]
4. Roest HIJ, Ruuls RC, Tilburg JJHC, Nabuurs-Franssen MH, Klaassen CHW, Vellema P, et al. Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands. Emerg Infect Dis. 2011;17:668–75 [PMC free article] [PubMed]
5. Tilburg JJHC, Rossen JWA, van Hannen EJ, Melchers WJG, Hermans MHA, van de Bovenkamp J, et al. Genotypic diversity of Coxiella burnetii in the 2007-2010 Q fever outbreak episodes in the Netherlands. J Clin Microbiol. 2012;50:1076–8 10.1128/JCM.05497-11 [PMC free article] [PubMed] [Cross Ref]
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8. Tilburg JJHC, Melchers WJG, Pettersson AM, Rossen JWA, Hermans MHA, van Hannen EJ, et al. Interlaboratory evaluation of different extraction and real-time PCR methods for detection of Coxiella burnetii DNA in serum. J Clin Microbiol. 2010;48:3923–7 10.1128/JCM.01006-10 [PMC free article] [PubMed] [Cross Ref]
9. Bleichert P, Hanczaruk M, Stasun L, Frangoulidis D. MST vs. IS1111 distribution: a comparison of two genotyping systems for Coxiella burnetii In: Proceedings of the 6th International Meeting on Rickettsiae and Rickettsial Diseases; Heraklion, Crete, Greece; 2011. Jun 5–7. p. 187.
10. Muskens J, van Engelen E, van Maanen C, Bartels C, Lam TJGM. Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA. Vet Rec. 2011;168:79–82 10.1136/vr.c6106 [PubMed] [Cross Ref]

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