Genotyping of Coxiella burnetii from domestic ruminants in northern Spain

Abstract

Background: Information on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST).

Results: A total of 45 samples from 4 goat herds (placentas, N = 4), 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20) and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21) were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM) samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several European countries, but some of the MLVA genotypes are described here for the first time.

Conclusions: Genotyping revealed a substantial genetic diversity among domestic ruminants from Northern Spain.

Figure 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 CP000733), RSA331 (CP000890), Nine Mile RSA493 (AE016828), CbuG Q212 (CP001019) and CbuK Q154 (CP001020) were determined in silico [13]using the published sequences. Each circle represents a unique genotype; the size of the circle corresponds to the number of samples with that genotype. Only full MLVA genotypes were included in this analysis. Branch labels and connecting lines correspond to the number of different markers between the genotypes. Genotypes connected by a gray background differ in only one marker from each other and may represent microvariants of one founder genotype. One cluster represents genotypes (I, J, M, AB and AC) obtained exclusively in cattle. The genotypes from cattle, goats and sheep (S, Z, AA and T) are clustered in two other groups.