Investigation of the Role of the European Brown Hare in the Epidemiology of Bacterial Zoonotic Pathogens: A Serological and Molecular Survey in Greece

Abstract

The aim of this study was to investigate the occurrence of Bartonella spp, Brucella spp, Coxiella burnetii, and Francisella tularensis in European Brown hares (Lepus europaeus) hunter harvested during 2-year hunting periods in northern and central Greece. Serum samples were examined for the presence of IgG antibodies by using an immune fluorescence test and/or an enzyme-linked immunosorbent assay. PCR was used to detect Bartonella spp DNA in blood samples and Brucella spp, C. burnetii, and F. tularensis DNA in liver samples. Antibodies against Bartonella spp were detected in 12 hares (12/105); whereas none of the hares examined was seropositive for Brucella spp, C. burnetii, and F. tularensis. The presence of Bartonella spp, Brucella spp, C. burnetii, and F. tularensis DNA was not detected in the samples examined. This study did not provide any evidence that the European Brown hare is involved in the epidemiology of Brucella spp, C. burnetii, and F. tularensis in Greece. However, our results suggest that this species is exposed to Bartonella spp, which gives the impetus for further investigation of its role as another host of this bacterium.

Introduction

Bartonella spp are facultative intracellular Gram-negative alphaproteobacterial (order Rhizobiales, family Bartonellaceae) distributed among a broad range of mammalian hosts, extending from humans to carnivores, ungulates, rodents, insectivores, bats, and lagomorphs (Vayssier-Taussat et al. 2009, Silaghi et al. 2016). Hematophagous arthropod vectors such as fleas, lice, sandflies, and ticks have been implicated in the transmission of bartonellae among different mammalian hosts (Bai et al. 2016, Silaghi et al. 2016).

Regarding lagomorphs, Bartonella alsatica was first isolated from the blood of wild rabbits in France (Heller et al. 1999). Since then, B. alsatica has been also detected in wild rabbits (Oryctolagus cuniculus) in Spain (Márquez 2010), and two human endocarditis cases and one human lymphadenitis case caused by B. alsatica have been reported (Raoult et al. 2006, Angelakis et al. 2008, Jeanclaude et al. 2009). B. alsatica DNA has been successfully detected in fleas (Spilopsyllus cuniculi) from wild rabbits in France and a European wildcat (Felis silvestris silvestris) in Spain (Márquez et al. 2009). Although S. cuniculi is a rare infestation in cats, they may get infected through close contact with rabbits and serve as a potential source of B. alsatica for humans. On the other hand, a study carried out in California did not detect bartonellae in blood specimens from riparian brush rabbits (Sylvilagus bachmani riparius) (Schmitz et al. 2014). Recently, the occurrence of Bartonella spp infection in the European Brown hare (EBH) was investigated in central Italy and based on the negative results obtained, the authors suggested that hares may not be very susceptible to these pathogens (Rocchigiani et al. 2018).

Among the Brucella spp considered highly pathogenic for domestic and wild animals, the EBH gets infected by Brucella suis biovars 1 and 2. Hares along with wild boars are important reservoir of B. suis biovar 2 (Godfroid et al. 2005, Gyuranecz et al. 2011a). However, hares can maintain B. suis biovar 2 and infect domestic animals (grazing pigs and cows) even in the absence of a wild boar population (Godfroid et al. 2005). Biovar 1 is highly pathogenic and causes severe disease in humans, whereas biovar 2 has only exceptionally been described as the causative agent of human brucellosis (Teyssou et al. 1989, Godfroid et al. 2005).

The wide range of Coxiella burnetii host species comprising arthropods, birds, and mammals suggests that complex reservoir systems may exist. Molecular and serological evidence of C. burnetii in hares has been previously reported and attributed to their feeding habits (Ejercito et al. 1993, Astobiza et al. 2011).

Francisella tularensis affects more than 300 species of mammals, especially lagomorphs and rodents (Moinet et al. 2016). In the terrestrial lifecycle, which is predominant in most European countries, the lagomorphs, terrestrial rodents, and ticks are the main source of human infections (Maurin and Gyuranecz 2016). The EBH is highly susceptible in F. tularensis and infection results in marked bacteremia, thus being a serious source of infection for blood-sucking ectoparasites. Moreover, their carcasses and excrements may contaminate the environment whereas those developing a chronic form of the disease become a permanent source of F. tularensis for other animals in the natural nidus as well as for humans (Treml et al. 2007).

Excluding F. tularensis, previous studies have shown the occurrence of the pathogens mentioned earlier in human and domestic animal populations in Greece (Tea et al. 2003, Diakou et al. 2017, Valiakos et al. 2017, Fouskis et al. 2018). However, the studies on wild animals and their involvement in the epidemiology of these pathogens are lacking. This study aimed at investigating the role of the EBH in the epidemiology of the important bacterial zoonotic pathogens Bartonella spp, Brucella spp, C. burnetii, and F. tularensis in northern and central Greece.

Materials and Methods

Hare samples

In total, samples from 105 free-ranging hares that were shot in northern (Central Macedonia and Eastern Macedonia and Thrace) and central Greece (Thessaly) over two consecutive hunting seasons (2012–2014) were included in this study; sera (n = 105), whole blood samples in EDTA (n = 49), and liver samples (n = 52) were collected by hunters and foresters according to the availability (Table 1). Blood samples were collected from the heart or the thoracic cavity of the shot hares. Blood samples without anti-coagulant were centrifuged, and sera were stored at −20°C pending examination for antibodies against Bartonella spp, Brucella spp, C. burnetii, and F. tularensis. Blood samples in EDTA and liver samples were stored at −20°C pending DNA extraction. Sera, blood, and liver samples were submitted still frozen to the Laboratory of Microbiology and Parasitology, University of Thessaly, Greece. Data on sex were recorded, when possible, for the subjects included in the study. The location data of hares were marked with handheld Global Positioning System Garmin units in the field.

Table 1.

Serum, Blood, and Liver European Brown Hare Samples Collected in Each Region of the Study Area

Region	Hare serum samples	Hare blood samples	Hare liver samples
Thessaly	31	24	27
Central Macedonia, northern Greece	38	15	15
Eastern Macedonia and Thrace, northern Greece	36	10	10
Total	105	49	52

Serological investigation

The serum samples (n = 105) were assayed for IgG antibodies against Bartonella spp, Brucella spp, C. burnetii, and F. tularensis by immune fluorescence test (IFAT). Fluorescein isothiocyanate conjugated sheep anti rabbit IgG (BioFX Laboratories, Owings Mills, MD) and commercial agent specific slides were used in the assay; IFAT slides with Vero cells infected with Bartonella henselae (Fuller Laboratories Fullerton, CA), both F. tularensis and Bartonella abortus antigens, each one placed in a separate spot (Fuller Laboratories Fullerton) and C. burnetii phase I and phase II antigens (Vircell, Granada, Spain). The starting dilution was 1:16 until the end-point titer. In the absence of previous serological studies in the EBH using IFAT for the detection of antibodies against B. henselae, Brucella spp, C. burnetii, and F. tularensis, the cut-off titers used in this study were based on serological studies in other domestic and wild animal species. For Bartonella spp, a cut-off titer for seroreactivity was defined as 1:64 (Fabbi et al. 2004, Goodman et al. 2005, Henn et al. 2007, Quinn et al. 2012, Schaefer et al. 2012, Diakou et al. 2017) and the endpoint titers were defined as the last dilution at which brightly stained organisms could be detected by fluorescence. The intensity of bacillus-specific fluorescence was scored subjectively from 1 to 4. Samples with a fluorescence score of ≥2 at a dilution of 1:64 were considered positive. The same two readers performed a double-blind reading of each slide. Titers ≥1:32 for C. burnetii (Pape et al. 2009a), B. abortus, and F. tularensis (Hotta et al. 2012) were considered positive. The negative and positive controls and the respective fluorescein isothiocyanate conjugates provided by the manufacturers were included on each slide.

The serum samples were also tested by using commercial indirect enzyme-linked immunosorbent assay (ELISA) kits for the detection of antibodies against C. burnetii (ID Screen^® Q Fever Indirect Multi-species, Grabels, France), which included wells coated with a C. burnetii phase I and II strain and Brucella spp (ID Screen Brucellosis Serum Indirect Multi-species, Grabels, France), which enables the detection of antibodies against B. abortus, Bartonella melitensis, and B. suis. In both ELISA kits, an anti-multi-species-IgG-HRP conjugate that recognizes mammalian IgG antibodies was included and used for the examination of the hare serum samples. Negative and positive controls provided by the manufacturer were used in the analysis.

Molecular investigation

Total genomic DNA extraction was performed by using a commercially available DNA extraction kit (Thermo Scientific GeneJET Genomic DNA Purification Kit; Thermo Fisher Scientific) in 49 blood and 52 liver hare samples. The purified DNA was stored at −20°C.

Bartonella 16S-23S ribosomal RNA intergenic spacer (ITS) PCR amplification, which amplifies a 604-bp fragment, was performed as previously described (Vissotto De Paiva Diniz et al. 2007) by using the primers 325s (5′-CTTCAGATGATGATCCCAAGCCTTYTGGCG-3′) and 1100as (5′-GAACCGACGACCCCCTGCTTGCAAAGCA-3′) on DNA extracts from 49 blood samples. Blood samples were further tested for the presence of Bartonella DNA by using a nested PCR approach (Rampersad et al. 2005) with primers P-bhenfa (5′-TCTTCGTTTCTCTTTCTTCA-3′) and P-benr1 (5′-CAAGCGCGCGCTCTAACC-3′), which amplifies an ∼186 bp fragment for B. henselae for the first amplification and nested primers Nbhenf1a (5′-GATGATCCCAAGCCTTCTGGC-3′) and Nbhenr (5′-AACCAACTGAGCTACAAGCC-3′), which amplifies an ∼152 bp fragment for B. henselae for the second round of amplification. One microliter of blood-extracted DNA template was used in the primary PCR reaction, and 1 μL of the primary reaction was used in the nested reaction.

The hare liver samples were examined by previously described conventional PCR assays for the detection of Brucella spp, C. burnetii, and F. tularensis DNA. Brucella 16S rRNA PCR amplification was carried out by using the primer pair F4 (5′-TCGAGC GCCCGCAAGGGG-3′) and R2 (5′-AACCATAGTGTCTCCACTAA-3′), which amplifies a 905-bp fragment, as previously described (Romero et al. 1995). The Trans-PCR assay was used for the detection of C. burnetii DNA with primers Trans1 (5′-TAT GTATCCACCGTAGCCAGTC-3′) and Trans2 (5′-CCCAACAACACCTCCTTATTC-3′) targeting a transposon-like repetitive region of the C. burnetii genome (Berri et al. 2000). The expected PCR product size was 687 bp. The 17-kDa gene PCR amplification with primers TUL4-435 (5′-GCTGTATCATCATTTAATAAACTGCTG-3′) and TUL4-863 (5′-TTGGGAAGCTTGTATCATGGCACT-3′) that yield a 400 bp fragment was performed for the detection of F. tularensis DNA (Johansson et al. 2000). Archived positive samples that were used as positive controls and negative controls without DNA were included in each PCR run. PCR products were visualized under UV-light, after 2% agarose gel electrophoresis and staining with ethidium bromide (0.5 μg/mL).

Results

Serological investigation

Antibodies against Bartonella spp were detected in 12 hare serum samples (12/105 [11.4%], 95% CI: 6.6–18.9), 11 samples from northern Greece (11/74 [14.8%], 95% CI: 8.5–24.6), and one sample (1/31 [3.2%], 95% CI: 0.5–16.1) from central Greece (Table 2). Titers of antibodies against Bartonella spp ranged from 1:64 to 1:128, serum titers of 1:64 in 11 hares, and 1:128 in one hare. None of the hare serum samples was found positive for antibodies against C. burnetii and Brucella spp by IFAT and ELISA and F. tularensis by IFAT.

Table 2.

Distribution of the European Brown Hare Seropositive Samples for Bartonella spp, Brucella spp, Coxiella burnetii, and Francisella tularensis

Region	Hare serum samples	Bartonella spp	Brucella spp	C. burnetii	F. tularensis
Region	Hare serum samples	Positive	Positive	Positive	Positive
Thessaly	31	1	0	0	0
Central Macedonia, northern Greece	38	4	0	0	0
Eastern Macedonia and Thrace, northern Greece	36	7	0	0	0
Total	105	12	0	0	0

Records on the sex were available for 39 out of the 105 subjects (17 males and 22 females). Out of the 12 Bartonella seropositive hares, sex was recorded for 5 individuals: 2 males and 3 females. The sex was unknown for the remaining seven seropositive individuals.

Molecular investigation

None of the blood samples examined was positive for Bartonella DNA in the 16S-23S ribosomal RNA intergenic spacer PCR amplification nor in the nested approach. Similarly, none of the examined hare liver samples was found to be positive for Brucella spp, C. burnetii, and F. tularensis DNA.

Discussion

This study reports the occurrence of Bartonella spp exposure of EBHs in northern and central Greece, with an overall seroprevalence of 11.4%. None of the hares examined was seropositive for Brucella spp, C. burnetii, or F. tularensis and PCR positive for any of the pathogens examined.

Serology can be used to infer previous exposure of a host to Bartonella spp. In this study, the occurrence of hare exposure to Bartonella spp is documented through the detection of IgG antibodies in 11.4% of the hare serum samples examined by using IFAT slides coated with cells infected with B. henselae. The seropositive results obtained are possibly justified by the cross-reactivity to common antigens among Bartonella spp (Chomel et al. 2004, MacDonald et al. 2004). A low level of cross-reaction with C. burnetii has been also reported in Bartonella infections (La Scola and Raoult 1996). However, none of the hares examined was seroreactive for C. burnetii in this study.

Because of cross-reactivity, bacterial isolation or PCR assays are necessary to identify the infecting Bartonella spp (La Scola and Raoult 1996, MacDonald et al. 2004). Further, enrichment culture of clinical specimens in an optimized, insect cell culture growth medium, before PCR testing, substantially increases the sensitivity of detecting Bartonella infection (Breitschwerdt 2017). Culture was not attempted in our study, as the blood samples were collected from the heart or the thoracic cavity of the shot hares by hunters and foresters; thus, the collection process was not strictly controlled, and it is not considered aseptic (Rampersad et al. 2005). Bartonella DNA failed to be amplified by using PCR in hare blood samples. Similar results were also previously obtained in central Italy where none of the examined EBHs was found to be positive for Bartonella DNA (Rocchigiani et al. 2018).

The occurrence of Bartonella spp in Greece has been documented by serological and molecular studies. A high antibody prevalence was found in free-roaming and stray cats (58.8%) (Diakou et al. 2017) as well as in humans (15–19.8%) (Tea et al. 2003). The molecular studies conducted so far concerned cats (8.5%) (Mylonakis et al. 2018), dogs (2 out of 50 dogs examined) (Diniz et al. 2009), and rodents (31.3% of the examined rodents, mainly Apodemus flavicollis) (Tea et al. 2004, Papadogiannakis et al. 2017). Although not being able to demonstrate the occurrence of Bartonella DNA in hare blood samples and to define the responsible species, our findings are suggestive of hare exposure to Bartonella spp in Greece as well.

Further studies are needed to infer the role of the hare in the transmission cycle of bartonellae. Although the detected antibody titers in this study were quite low, the presence of antibodies against Bartonella spp indicates exposure to the pathogen possibly after ectoparasite infestation. Hares are often infested with ectoparasites, including fleas, ticks, and lice (Dik and Uslu 2018). The EBH is a secondary host of S. cuniculi that is widely distributed after its primary host, the rabbit, and it has been implicated in the transmission cycle of B. alsatica in wild rabbits, cats, and humans. Moreover, competent vectors of Bartonella spp such as the flea species Pulex irritans and Nosopsyllus fasciatus have been detected in the EBH (Dik and Uslu 2018). Among the tick genera reported in hares, Ixodes, Haemaphysalis, and Dermacentor (Dantas-Torres et al. 2011, Psaroulaki et al. 2014, Dik and Uslu 2018) were found to be infected by several Bartonella spp (Sréter-Lancz et al. 2006, Breitschwerdt 2017) in previous studies. The results obtained so far support the role of the EBH in the eco-epidemiology of Bartonella spp as a maintainer or carrier of infected vectors and not as an alternative reservoir. This species, while keeping its small home range, shares the same ectoparasites and habitat with other domestic and wild animals, some of which may be important hosts of Bartonella spp or even reservoirs of zoonotic bartonellae such as cats and rodents (Marié et al. 2006, Chomel and Kasten 2010, Silaghi et al. 2016).

B. melitensis is the predominant species responsible for human and animal brucellosis in Greece. Despite the policies followed in Greece, brucellosis remains endemic while presenting one of the highest incidences (1.43 cumulative incidence per 100,000 of population based on the cases declared from 2007 to 2012). Notably, the division of Thessaly appears to have the highest incidence (4.87/100,000) whereas an increased incidence in the division of Eastern Macedonia and Thrace was recorded due to the 2008 Thasos epidemic. As for small ruminants, the total percentage of positive farms in Greece was 4.80%. From the regions under the vaccination program, the highest prevalence value was detected in Larissa, Thessaly (34/273; 12.45%) (Fouskis et al. 2018). Regarding the seroprevalence of B. suis, a serosurvey conducted previously in swine herds in northern and southern Greece showed a 3% seroprevalence (Burriel et al. 2003). The absence of antibodies against Brucella spp in the EBH found in this study is consistent with previous studies conducted in the Czech Republic (Hubálek et al. 1993) and Germany (Frandölich et al. 2003), suggesting that Lepus europaeus plays no significant role as a wildlife reservoir for B. suis in Greece. However, up to 17% seroprevalences for Brucella spp were reported elsewhere in hares (Winkelmayer et al. 2005).

The seroprevalence of C. burnetti in the human population in northern Greece was reported to be 7.5% (Pape et al. 2009b); whereas 35% of the dairy cattle herds examined in central and northern Greece (Dovolou et al. 2011), 6.5% of goats and 10.4% of sheep in northern Greece (Pape et al. 2009a), and 14.5% of sheep and 15% of goats in central Greece (Valiakos et al. 2017) were found to be seropositive. Our findings on the absence of antibodies against C. burnetii in the EBH are consistent with studies previously conducted in the Czech Republic (Hubálek et al. 1993) and Germany (Dedek et al. 1990). A possible explanation for the negative results of our study is that the hare population examined did not get infected when fed on pastures that semi-extensive livestock animals use in summer and could possibly contaminate them with C. burnetii positive feces (Ejercito et al. 1993).

Serological tests are useful diagnostic tools for epidemiological surveys of tularemia in EBHs as they seroconvert, and they can potentially carry viable bacteria over a longer time span and thus serve as a reservoir species (Gyuranecz et al. 2011b). Previous studies in other European countries showed that the seroprevalence of F. tularensis in hares ranges from 4.5% to 6.5% (Treml et al. 2007). To our assumption, there are no studies on F. tularensis in Greece. The negative serological findings in the hare population from this study are consistent with the existing knowledge that Greece is among the European countries that are deemed to be free of tularaemia (Maurin and Gyuranecz 2016).

Conclusions

Our findings do not support the role of the EBH in the epidemiology of Brucella spp, C. burnetii, and F. tularensis in Greece and they are suggestive of EBH exposure to Bartonella spp. The studies conducted so far support the lack of hare competence as a reservoir host for Bartonella spp. It is possible that the hare acts as a maintainer or carrier of infected vectors, but this hypothesis remains to be confirmed by other studies. The need for culture or enrichment culture of clinical specimens before PCR testing and aseptic sampling is highlighted here and should be considered in future studies to draw solid conclusions on the presence or absence of Bartonella DNA in clinical samples.

Ethics

The hare serum, blood, and liver samples included in the study represent material collected opportunistically (no active capture, killing, and sampling of wild animals specifically for this study was performed) from animals hunter-harvested by members of the Greek Hunting Federation of Macedonia and Thrace and the Greek Hunting Federation of Thessaly and Sporades, during the hunting seasons, according to the prerequisites of the Greek Legislation and submitted to our laboratory for Passive Wildlife Disease surveillance. Thus, special approval was not necessary and steps to ameliorate suffering were not applicable in this study. Research on animals as defined in the EU Ethics for Researchers document (European Commission 2007, Ethics for Researchers—Facilitating Research Excellence in FP7, Luxembourg: Office for Official Publications of the European Communities, ISBN 978-92-79-05474-7) is not a part of the study.

Footnotes

Acknowledgments

The authors gratefully acknowledge the foresters and the hunters of the Hunting Federation of Macedonia and Thrace and the Hunting Federation of Thessaly and Sporades for providing the hare samples that were included in this study.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

No funding was received for this article.

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