Surveillance and outbreak reports Q fever epidemic in Hungary , April to July 2013

M Gyuranecz (m.gyuranecz@gmail.com)1,2, K M Sulyok1,2, E Balla3, T Mag3, A Balázs3, Z Simor4, B Dénes5, S Hornok6, P Bajnóczi4, H M Hornstra7, T Pearson7, P Keim7, A Dán5 1. Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary 2. These authors contributed equally to this article 3. National Center for Epidemiology, Budapest, Hungary 4. Government Office for Baranya County, Pécs, Hungary 5. Veterinary Diagnostic Directorate, National Food Chain Safety Office, Budapest, Hungary 6. Faculty of Veterinary Science, Szent István University, Budapest, Hungary 7. Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, United States


Introduction
Coxiella burnetii is the causative agent of Q fever, a worldwide zoonotic disease [1,2].Domestic ruminants are the most important recognised reservoirs of C. burnetii; they are often asymptomatic carriers, but the agent may also cause abortion in these animals [1,3].Ticks may also act as reservoirs of C. burnetii in nature [1,4].Domestic ruminants are considered the most important source of human Q fever infection.Outbreaks in human populations have been linked to slaughterhouses or dispersion of C. burnetii by wind from farms where infected ruminants were kept [1,5].Q fever is typically an acute febrile illness with nonspecific clinical signs such as atypical pneumonia and hepatitis in roughly 40% of cases, while 60% remain asymptomatic after infection [6].A small percentage (ca 5%) of infected people may develop chronic infection with life-threatening valvular endocarditis [7,8].
Q fever is a notifiable disease in Hungary.Antibodies against C. burnetii were first detected in the sera of abattoir workers in 1950 [9], and infections were first diagnosed in 1956 in dairy and sheep farms [10].The last major outbreaks were registered in the period of 1976-80.According to a recent survey (2010-11), seroprevalence among cattle and sheep in Hungary were 38.0% and 6.0%, respectively, by enzyme-linked immunosorbent assay (ELISA), which is in agreement with the European averages [3,11].The number of yearly reported acute human infections in Hungary ranged between 36 and 68 between 2008 and 2012.
The aim of this study was the epidemiological, diagnostic and genetic investigation of the Q fever outbreak that occurred in Hungary during the spring and summer of 2013.

Methods
During the epidemic investigation a suspected human case was defined by high fever and radiological evidence of pneumonia occurring after 17 April 2013, and by geographic proximity to the outbreak area after 4 April.The human serum samples were tested with micro-immunofluorescence test (MIF) (Focus Diagnostics, United States).Based on MIF results of the first serum samples, the suspected human cases were evaluated as seronegative or possibly infected (showing seropositivity, i. e. any phase of specific IgM/ IgG antibodies).Cases were considered confirmed if there was seroconversion and/or an elevated level (greater than 1:252 dilution) of IgG-II/IgG-I antibodies when paired sera were available.DNA was extracted from EDTA anticoagulated human blood and serum samples (collected in the one to three weeks after onset of clinical symptoms) using the MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics, Switzerland).
Animal serum samples were tested with complement fixation test (CFT) (Virion/Serion GmbH, Germany) and ELISA (IDEXX Laboratories Inc., Switzerland).Questing ticks were collected using the dragging/flagging method.DNA was extracted from milk, manure and individual tick samples with the ReliaPrep gDNA Tissue Miniprep System (Promega Inc., United States).
All extracted DNA samples were screened with a realtime PCR assay targeting the IS1111 element of C. burnetii [12].Strong positive samples (with Ct values below 30) were further genotyped with multispacer sequence typing (MST) based on 10 loci and multilocus variablenumber tandem repeat analysis (MLVA) based on six loci [13][14][15].

Description of the epidemic area
The centre of the outbreak region was a hilly area of approximately 10 km 2 encompassing primarily the small rural towns of Vokány (851 population) and Kistótfalu (321 population) of Baranya county in southern Hungary (45.90 °N, 18.33 °E) (Figure 1).Although the area has a continental climate, an unusually cold and snowy March was followed by a warm, dry and occasionally windy April in 2013.A non-dairy merino sheep flock (450 ewes) near Vokány, a small cattle herd (40 animals), a small mixed flock of sheep and goats (20 animals) and some individually kept animals were the only livestock in the region.The lambing season occurred from January to April in the area in 2013 (Figure 2).

Investigation of human cases
During systematic screening for respiratory pathogens, six patients of the initial cluster of eight febrile cases (since 17 April) showed C. burnetii-specific phase II IgM  A three-week course of doxycycline therapy (200 mg daily) was administered to all laboratory-confirmed cases and also to the majority (no exact data are available) of the suspected cases living in the outbreak area.Serological follow-up was advised for all patients diagnosed with acute Q fever to control their IgG-I level because persisting high titres (≥800) may indicate chronic infection.As of July 2014, third serum samples have been sent from 21 of the 70 confirmed cases, and high titres of IgG2/IgG1 (≥512) were detected in all of them.

Animal and environmental investigations
Fifty-six serum and 20 milk samples were collected from the Vokány merino sheep flock and from all animals on other farms.Manure samples were also collected from the breeding stable of each farm within the outbreak region.

Figure 2
Timeline of the epidemiological and diagnostic investigations during the Q fever outbreak in Hungary, 2013 Columns: number of laboratory-confirmed male (blue) and female (red) cases according to the date when first serum samples were taken.The CFT and ELISA examinations of the serum samples collected in the merino sheep flock revealed 23.2% (13/56) and 44.6% (25/56) seropositivity, respectively, while C. burnetii was detected in four of 20 (Ct: 30.1-33.5) of individual's milk and two thirds (41/65, Ct: 28.9-36.82) of manure samples collected from the merino sheep flock.Of the four sheep with PCR-positive milk, two were also seropositive by ELISA.The ELISA showed 11 of 40 and two of 20 seropositive animals in the cow herd and the mixed sheep and goat flock, respectively, and only one of the milk samples from a cow contained C. burnetii DNA (Ct: 33.7).The manure samples collected from these small farms as well as all samples (serum, milk, manure) collected from the individually kept animals in the epidemic region were negative for C. burnetii infection.

Genetic characterisation of C. burnetii strains
The MST examination of C. burnetii DNA detected in one human sample and two manure samples from the merino sheep flock revealed sequence type (ST)18.The MLVA pattern of the sheep and human strains were also almost identical, 4/5-9-3-3-0-5 (Ms23-Ms24-Ms27-Ms28-Ms33-Ms34).Genotyping of samples from the ticks collected in 2011 and from the cow milk failed because of their low C. burnetii DNA content.

Discussion
Seventy human cases were confirmed during the Q fever outbreak in Hungary in 2013.The laboratory diagnosis of the first patients based on serology and real-time PCR identified the aetiologic agent within a few days and enabled targeted screening and adequate therapy of further infected individuals.Combining these two methods was also an effective diagnostic strategy in the first two weeks after the onset of clinical symptoms.The merino sheep flock in Vokány village was identified as the source of the outbreak.Interestingly, the farmer had not observed an elevated abortion rate during the lambing season from January to April.The 44.6% seropositivity rate at this farm was far higher than the 6.0% recorded in other Hungarian sheep flocks and represents strong evidence of a localised outbreak [11].
The causative agent of the outbreak described here was an ST18 C. burnetii strain, which argues against a direct connection with the 2007-09 outbreak in the Netherlands which was caused primarily by ST33 [16,17].ST18 has previously been detected in France, Germany, Italy, Romania and Slovakia [13].A recent human serum sample from Belgium was also likely to be (or be closely related to) ST18 [17].Our subtyping data confirmed that the source of the human Q fever infections was the merino sheep farm.The one VNTR difference between the ovine and human isolates at Ms23 is likely to be due to the rapid mutation capacity of this locus [15].We hypothesise that dried manure and birth fluids contaminated with C. burnetii were dispersed by the wind from the sheep farm towards the towns and their inhabitants.This hypothesis is supported by the genotyped human sample.This patient had not had any direct contact with the merino sheep flock in Vokány.He was an inhabitant of a neighbouring town but was working on a hillside close to Vokány at the time of his supposed exposure.Based on the available epidemic information, laboratory data and the large number of human cases it is highly unlikely that this epidemic was caused either by tick-bites or raw milk consumptions.The origin of the ST18 strain at the merino sheep farm remains unknown.C. burnetii isolates originating from other parts of Hungary have been either ST20 (cattle) or ST28 and ST37 (sheep) genotypes [18].
Public health countermeasures included elimination of manure in June 2013, followed by disinfection of the merino farm (using VIROCID, Cid Lines Inc, Belgium and Disinflex, Hexachem Kft, Hungary) in July.In this region, no further acute human Q fever cases have been confirmed since July 2013.Manure and milk samples collected in May 2014 from the merino sheep flock in Vokány and other farms within a 30 km diameter, tested negative by real-time PCR.As a precaution, the merino farm in Vokány was disinfected in June 2014 as well.

Figure 1
Figure 1Geographic expansion of the Q fever epidemic in Hungary, 2013 Ixodes ricinus and two Haemaphysalis concinna) were collected from the vegetation at the merino sheep farm pasture in July and September 2013.Another 115 archived ticks collected from two dogs (four Dermacentor marginatus) and 23 goats (91 I. ricinus and 20 H. concinna) residing with the merino sheep flock in 2011 and 2012 were also included in this study.C. burnetii DNA was detected in five archived I. ricinus (three larvae, one nymph, one female, Ct: 35.0-36.58)collected from goats kept in the merino sheep flock in 2011.