First serological record of Coxiella burnetii infection in the equine population of Slovakia

Coxiella burnetii is a worldwide zoonotic pathogen causing Q fever in various animal species and humans. In Slovakia, cases of C. burnetii infection in both animals and humans are confirmed every year. The role of horses in the epidemiology of this neglected disease is still unclear. In our study, we focused on a serosurvey of C. burnetii in the equine population in Slovakia by the ELISA method. Subsequently, a nested PCR was performed to detect the 16S rRNA fragment of the genus Coxiella. Among 184 horse sera, the presence of specific antibodies to C. burnetii was detected in four samples, representing a 2.17% seropositivity. All the positive horses were mares; two originated from Central Slovakia and two from Eastern Slovakia. Although the number of positive samples was too small for a determination of statistical significance, our results provide the first confirmation of antibodies to C. burnetii in horses from Slovakia. Although no positive PCR result was obtained, these serological findings may help to clarify the circulation of the pathogen in the environment.


Introduction
Q fever is a worldwide spread zoonotic disease associated with severe illness in humans and animals. Coxiella burnetii (Derrick, 1939) is a bacterium that can infect a wide range of animals, e.g. sheep, goats, cattle, dogs, cats, horses, birds, rodents, and ticks. The infected animals shed C. burnetii in their birth products, faeces, milk, and urine. However, the circulation dynamics of C. burnetii in and through horses is still unclear. The natural reservoir of C. burnetii encompasses many free-living vertebrates, but the major risk of infecting humans arises through contact with infected ruminant livestock and their contaminated products, mainly through the inhalation of contaminated aerosols of birth fluids either from abortions or from normal parturitions (Aitken 1989;Honarmand 2012;Roest et al. 2013a;Aljafar et al. 2020;Khademi et al. 2020;Abdel-Moein and Zaher 2021). The organism leads an obligate intracellular life cycle, during which it multiplies in the phagolytic compartments of the phagocytic cells in the host s immune system. This characteristic makes studying the organism particularly difficult, and it is perhaps one of the reasons why much still remains unknown about the organism and its pathogenesis (Bewley 2013). Coxiella burnetii is frequently detected in ticks and laboratory experiments have revealed that at least some tick species are competent vectors. Coxiella burnetii has been considered to be the only species of the Coxiella genus. However, there is evidence that Coxiella-like (CL) organisms have a high homology with the pathogenic C. burnetii, based on the 16S rRNA sequence phylogenetic analyses (Gottlieb et al. 2015;Trinachartvanit et al. 2018), and they are widespread in ticks (Rahal et al. 2020;Chisu et al. 2021). In Slovakia, nonpathogenic CL microorganisms have been registered in ticks (Špitalská et al. 2018). Although some CL endosymbionts of ticks may not play any role in inducing pathology, the possibility of a CL microorganism transformation leading to the emergence of Q fever has been noted (Duron et al. 2015a). Thus, ticks may represent potential sources of Q fever infection for humans and animals (Knap et al. 2019). In Slovakia, the presence of C. burnetii was previously described in Ixodes ricinus (Linnaeus, 1758), Dermacentor reticulatus (Fabricius, 1794), Dermacentor marginatus (Sulzer, 1776), Haemaphysalis concinna (Koch, 1844), and Haemaphysalis inermis (Birula, 1895) ticks (Řeháček et al. 1991;Špitalská and Kocianová 2003;Špitalská et al. 2018). Slovakia is a country with a dense tick population due to a high abundance of various biotopes for ticks, which range from meadows and forests of the Pannonian lowlands to the Carpathian woods. The diversity of habitats and a wide range of vertebrate hosts (birds, small mammals, deer and wild boar) create suitable conditions for the survival of ticks (Kocianová et al. 2008). An infection caused by C. burnetii is often difficult to identify due to its inapparent course, but abortions and infertility have been described in horses (Marenzoni et al. 2013). Stillbirths and neonatal mortality were also recorded in horses in association with C. burnetii (Acland 1993).
The aim of our study was to confirm the circulation of the neglected pathogen, C. burnetii, in the equine population of Slovakia using a serological analysis and a subsequent PCR detection.

Sample collection
A total of 184 healthy horses (97 mares, 21 stallions and 66 geldings) from the Western, Central and Eastern regions of Slovakia were investigated (Table 1). Blood, with and without anticoagulants, was collected from the jugular vein in the period from April 2018 to October 2020 for PCR and serological analyses. The samples were obtained from 16 various, randomly-selected locations in Slovakia ( Fig. 1).

Serological analysis
The presence of IgG antibodies to C. burnetii in the serum samples was investigated by the ELISA method using the ID Screen Q Fever Indirect Multi-species Kit (IDvet, Montpellier, France). The procedure was performed according to the manufacturer's instructions. The results were expressed as an optical density ratio of the sample to the positive control (S/P) based on the following calculation: The samples with a S/P less than 40% were determined to be negative, and for the purpose of the present study, doubtful samples (S/P values between 40% and 50%) were considered negative; while the samples with a S/P value more than 50% were evaluated as positive.

DNA isolation and PCR analysis
Genomic DNA was isolated from whole blood using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The isolated DNA was stored at −20°C until it was used.
The 16S rRNA gene of the Coxiella genus was amplified by a modified nested PCR, as described by Seo et al. (2016). The first round of the PCR was performed using specific primers Cox16S-F1 (5´-CGTAGGAATCTACC TTRTAGWGG-3′) and Cox16S-R (5´-GCCTACCC GCTTCTGTACAATT-3′) with an amplicon size of 1321-1429 bp. For the second round, the primers Cox16S-F2 (5´-TGAGAACTAGCTGTTGGRRAGT-3′) and Cox16S-R were used for the amplification of the

Results
Our results (Table 1) confirmed a 2.17% seroprevalence of antibodies to C. burnetii in the horses in Slovakia. All of the horses were without any clinical symptoms of Q fever at the sampling time, as well as three months earlier. However, tick bites had been noted on all the horses by their owners in the past. Specific IgG antibodies were detected in four mares. One animal belonged to the age group "younger than or equal to 10 years" and three mares were "older than 10 years". In association with the localisation, we confirmed two positive cases at the same stable in Central Slovakia, and two positive cases, also at the same stable, in Eastern Slovakia. No statistical analysis was performed, as the size of the positive sample was too small. No blood sample was PCR positive for the 16S rRNA of the Coxiella genus.

Discussion
The results summarised in this study represent the first information about the prevalence of IgG antibodies to C. burnetii in the sera of horses bred in the territory of Slovakia. Out of 184 examined horses, four (2.17%) animals were seropositive. All of the positive samples originated from mares. Three seropositive mares were older than 10 years. To date, the presence of C. burnetii in Slovakia has been reported in different species, including ticks, birds, and sheep (Řeháček et al. 1991;Špitalská and Kocianová 2003;Dorko et al. 2010;Berthová et al. 2016). The presence of antibodies to C. burnetii has also been confirmed in the captive breeding of wild animals, such as mouflons, goats, sheep and fallow deer kept at ZOO Košice (Dorko et al. 2009). Human cases of Q fever are reported in Slovakia where the notification rate in humans was 0.02 per 100,000 in 2019, and 0.04 per 100,000 population in 2018 (one case in 2019, two cases in 2018; and no human case was reported in 2015-2017) (EFSA andECDC 2019, 2021). In Slovakia, a 4.07% seroprevalence of antibodies to C. burnetii in cattle was also reported (MARDSR 2019).
The particular role of horses as reservoirs of C. burnetii in Slovakia has not yet been determined. However, there are indications that horses may play an important role in the spread of Q fever as reservoirs for C. burnetii (Khademi et al. 2020). Seo et al. (2016) described antibodies to C. burnetii in the horses of South Korea. They identified 11 samples out of 816 horses (1.3%) as being seropositive for C. burnetii by ELISA. Also, Desjardins et al. (2018) described antibodies to C. burnetii in the horse sera in endemic areas of Fig. 1 Sampling locations; number of horses coming from Slovakia; the green colour represents the occurrence of seropositive horses and yellow colour represents the total number of sampled horses in the stables Southeast France, in Camargue (west of the mouth of the Rhône River) and on the Plain of La Crau (east of the mouth of the Rhône River). They confirmed a 4% and 12% seroprevalence in 2015 and 2016, respectively. Furthermore, they confirmed an association between the locations with seropositive horses and those where Q-fever-related human cases were previously described. Li et al. (2020) confirmed by PCR the presence of C. burnetii in 39.5% of the investigated horses in China. Unlike these researchers, in our study we failed to detect and molecularly characterise the pathogen in horses. The PCR negativity may have been due to the fact that the horses did not suffer from an acute infection at the time of the sampling. However, the seropositivity of some animals indicates previous contact with the pathogen or even a past infection.
Some literary sources report clinical Q fever cases in a community of horseback riders or people visiting horse facilities (Nett et al. 2012;Roest et al. 2013b). In addition to direct contact with infected horses, ticks infesting horses may also represent a risk factor in the Q fever transmission (Roest et al. 2013b;Duron et al. 2015b;Desjardins et al. 2018). The presence of C. burnetii was detected in aborted equine placentas and foetuses (Leon et al. 2012;Runge et al. 2012). All the seropositive horses examined in the present study were mares, but the history of abortions in these animals is not known. Although no transmission of infections from horses to humans has been reported, studies have hypothesised that some individuals, such as equine veterinarians or breeders, could potentially be at a higher risk of infection (Karagiannis et al. 2009;Palmela et al. 2012;Van den Brom et al. 2013;Sun et al. 2016;Akter et al. 2020).
Until now, only limited facts about the epidemiology of C. burnetii in horses has been available. Marenzoni et al. (2013) indicated a possible role of horses as sources of the pathogen for other animal species, as well as humans. Similarly, Roest et al. (2013b) and Seo et al. (2016) stated that horses are probably the reservoirs of C. burnetii for other susceptible organisms. Our serological survey provides the first evidence to date of seropositivity to C. burnetii in the horse population of Slovakia. This could be the basis for further research on this neglected, but serious vector-borne zoonosis.

Declarations
Ethic statement The study was performed according to the guidelines of The Ethics Committee of the University of Veterinary Medicine and Pharmacy in Košice. All animal samples examined in this study were collected by veterinarians upon the consents of the animal owners.

Conflict of interest There is no conflict of interest.
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