The temporal and spatial diversity of Coxiella burnetii genotypes associated with human and animal disease in Portugal was analysed using a 6-locus multiple-locus variable-number tandem repeat analysis (MLVA) and a 10-locus multi-spacer sequence typing (MST) panel. Fifteen cultured C. burnetii isolates from 13 Q fever patients and a stillborn goat and 6 additional PCR-positive ruminant tissue samples obtained during 2006-2011 were included in this study. Seven MLVA genotypes (types S-Y) were obtained, including 4 new MLVA types (U, V, W, and X), all corresponding to 3 MST profiles (types 4, 8, and 13) previously reported from France and Spain. MLVA types U-Y, all belonging to MST type 4, were found in acute Q fever patients from the districts of Évora, Faro, Lisbon, and Setúbal. Different MLVA types were associated with goats from Castelo Branco district (S) and chronic Q fever patients from both Castelo Branco and Lisboa districts (S and T), matching with MST types 13 and 8, respectively. In conclusion, a genotypic diversity of C. burnetii consistent with a non-outbreak situation was identified. The involvement of different genotypes in acute and chronic Q fever was found, linking one of the chronic genotypes to goats from the eastern region of the country.

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nesium chloride (Promega), 0·5 U GoTaq Flexi DNA polymerase (Promega) and 2·5 µl of sample DNA. C burnetii Nine Mile reference strain DNA was used as the positive control. Amplification was performed using a modified touchdown PCR program (Berri and others 2000); 10 µl of the amplification products were analysed in 1·5 per cent agarose gel electrophoresis stained with ethidium bromide and photographed under a UV transilluminator. Restriction endonuclease analysis with AluI (Promega) was performed in a 20 µl reaction volume for 90 minutes at 37°C, using 5 µl of the PCR amplification products. The digested products were analysed by agarose gel electrophoresis, as described above. The specific 687 base pair (bp) PCR amplification product and the 316, 191 and 179 bp fragments obtained by restriction endonuclease analysis allowed the specific identification of C burnetii (Fig 1).The presence of C burnetii DNA in all the samples tested, and the absence of other aetiological agents, support the hypothesis that C burnetii infection was responsible for the abortion in the waterbuck and the stillbirth in the antelope.C burnetii is known to be widespread in the environment and to have a large number of possible reservoirs. Ticks are also considered to be a major reservoir and vector

Coxiella burnetii is a non-motile, obligate intracellular, Gram-negative microorganism that is the causative agent of Q fever, a widespread zoonosis. Cattle, sheep and goats are considered to be the primary sources of human infection (McQuiston and Childs 2002), although occasional zoonotic infections have been linked to pets, birds, ticks and other arthropods (Buhariwalla and others 1996, Maurin and Raoult 1999). Human beings are infected by the inhalation of contaminated aerosols or the ingestion of infected fresh milk and dairy products. The disease in human beings is usually asymptomatic or characterised by a febrile syndrome with spontaneous recovery. In the acute form, pneumonia and granulomatous hepatitis may occur; endocarditis is the predominant clinical manifestation of chronic cases (Maurin and Raoult 1999). In animals the infection is generally subclinical, although abortions at the end of pregnancy, stillbirths and the delivery of weak offspring, and cases of retained placenta, endometritis, infertility and low birth rates may occur (McQuiston and Childs 2002, Arricau-Bouvery and Rodolakis 2005). Q fever is rarely reported and infections in livestock often go unnoticed; if sporadic abortions occur, they may not prompt an investigation. Infection is usually identified when human beings are affected through zoonotic exposure. Little is known about the occurrence and significance of C burnetii infection in domestic ruminants in Portugal. The infection has been reported sporadically in human beings, domestic ruminants, zoo animals (Clemente and others 2008) and companion animals (Bacellar and others 1995). The aim of the present study was to detect C burnetii DNA in fetal tissues, placentas and vaginal discharges from livestock that aborted naturally, and to gain evidence of its role in the occurrence of abortions. Between October 2006 and March 2008, 91 samples, from 29 cattle, 25 sheep and 37 goats with a history of abortion, were submitted to the authors' laboratory to investigate whether they were infected with C burnetii. The farms of origin were located in the northeastern and eastern mountainous regions, and in the south and southeast of Portugal. The samples were four vaginal swabs, 10 placentas and 77 pools of organs (brain, liver, spleen and lung) collected from the aborted fetuses (Table 1). The diagnosis of C burnetii was confirmed by PCR.