1 Molecular survey on the occurrence of arthropod-borne pathogens in wild brown hares (Lepus europaeus) from Central Italy
Guido Rocchigiani, Valentina Virginia Ebani*, Simona Nardoni, Fabrizio Bertelloni, Alice Bascherini, Alessandro Leoni, Francesca Mancianti, Alessandro Poli
Department of Veterinary Science, University of Pisa, viale delle Piagge 2, 56124 Pisa, Italy
*Corresponding author:
Valentina Virginia Ebani, DVM, PhD Department of Veterinary Science University of Pisa
Viale delle Piagge 2, 56124, Pisa, Italy
valentina.virginia.ebani@unipi.it
Authors:
Guido Rocchigiani guido.rocchigiani.g@gmail.com
Simona Nardoni simona.nardoni@unipi.it
Fabrizio Bertelloni fabrizio.bertelloni@vet.unipi.it
Alice Bascherini aliwonder@email.it
Alessandro Leoni alessandro.leony@libero.it Francesca Mancianti francesca.mancianti@unipi.it
2 Abstract
Data about the spreading of arthropod-borne pathogens among hare populations are very scant, so the aim of the present preliminary study was to investigate, through molecular analysis, the occurrence of Anaplasma phagocytophilum, Bartonella sp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Francisella tularensis, Leishmania spp. and piroplasms DNA in blood of 51 wild hares (Lepus europaeus) living in protected areas in Tuscany. All hares resulted negative for A. phagocytophilum, Bartonella sp., B. burgdorferi s.l., C. burnetii and F. tularensis. Five animals (9.8%) were positive for L. infantum.Leishmania and o One hare (1.9%) tested positive for piroplasms. Sequencing of this sample showed a piroplasm similar but not identical to .already one Babesia isolate from the same animal species described in Turkey. Therefore, to the best of our knowledge, this is the first molecular report of piroplasms occurring in wild hares from Italy, and the second worldwide.
The examined hares appeared to be in good health status, corroborating the hypothesis of a chronic carrier state of some vector borne agents this animal species.
Babesia belonging to the clade I and comprehending most species of Babesia sensu stricto.
Key words: Hares; Arthropod-borne pathogens; Babesia sp.; Leishmania spinfantum
1. Introduction
The brown hare (Lepus europaeus Pallas, 1778) is widely present in Europe, including Italy. In the past years the wild hares population of Central Italy considerably decreased, but successively, about twenty years ago, these lagomorphs have been reintroduced from East Europe, mainly to restock protected and hunting areas.
Wild hares may be exposed to different hematophagous arthropods, such as ticks, mosquitoes, sandflies and fleas, that often are vectors of several pathogens, comprehending zoonotic agents. Anaplasma phagocytophilum is an obligate intracellular zoonotic bacterium able to infect several domestic and wild animal species. It is transmitted through ticks’ bite, in particular Ixodes ricinus. Small mammals, such as voles and mice, have been suggested as reservoir of this pathogen, whereas the role of wild lagomorphs is not fully understood (Hulinská et al., 2004).
Bartonella species are facultative intracellular bacteria, mainly transmitted by hematophagous arthropods, responsible for mild and severe clinical forms in animals and humans. Bartonella infections have been detected in asymptomatic animals too, in particular wild rodents that are considered the main reservoirs (Silaghi et al., 2016).
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3 Several bird species and small mammals including rodents, shrews, hedgehogs and hares are considered the reservoirs of Borrelia burgdorferi sensu lato (s.l.), agent of Lyme disease in humans and animals (Lindgren and Jaenson, 2006).
Coxiella burnetii is a zoonotic bacterium traditionally related to domestic ruminants in which determines reproductive disorders. Even though a sylvan cycle involving wild animals and hematophagous arthropods is well known, data about the spreading of C. burnetii among hares are scant (Psaroulaki et al., 2014).
Lagomorphs are considered the main reservoir of Francisella tularensis. In these animals the agent causes acute septicemia or subacute-chronic disease with necrosis in livers, spleen, lung and bone marrow. Humans can acquired the infection by direct contact with infected animals, through inhalation of contaminated aerosol or ingestion of contaminated water and/or uncooked infected game. Transmission through bites by infected hematophagous arthropods are possible too (Runge et al., 2011).
Babesia and Theileria are two genera of Apicomplexan protozoa, commonly indicated as Piroplasms. They are both transmitted by ticks and able to infect red cells and/or leucocytes of mammals. Piroplasmosis is characterized by different clinical signs, comprehending fever, anaemia and jaundice (Schnittger et al., 2012). In hares the occurrence of clinical piroplasmosis is considered very rare (Baldelli, 1960; Vaccari et al, 1961) and only a few asymptomatic infections have been reported worldwide in this species. At the best of our knowledge, only two Babesia species from Turkey have been molecularly characterized (Orkun and Karaer, 2017)..
Leishmania spp. are flagellate protozoa, transmitted by phlebotomine sandfly species. In Mediterranean area dogs are the major reservoir hosts of Leishmania infantum; Iberian hares (Lepus granatensis) were recently found infected by this agent and proven to be infective for Phlebotomus perniciosus, the main cyclic vector in Europe (Molina et al., 2012). Furthermore hares have in fact been considered responsible for the outbreak of human leishmaniasis, affecting metropolitan Madrid (Jimenez et al., 2013) and were reported as preferred blood meal by sandflies (Gonzalez et al., 2017).
Wild hares, if infected by zoonotic agents, may be source of infection for humans, particularly hunters during the carcasses manipulation. Moreover, hares can favorite the spreading of arthropods harboring pathogens able to infect other animals, other than people.
In view of these considerations and being the above reported pathogens scarcely investigated in wild hares populations in Italy, aim of the present study was to evaluate the occurrence of A. phagocytophilum, Bartonella sp., B. burgdorferi., C. burnetii, F. tularensis, Leishmania spp. and piroplasms in wild hares captured in protected areas in Tuscany.
4 2. Material and methods
2.1 Animals
Blood samples were collected from 51 wild brown hares captured in three different protected areas of the province of Pisa (Central Italy; 43°43’N – 10°24’E) with nets during 2016-2017 winter (average temperature 6.9° C). In a previous survey for the same areas a population density ranging from 2 to 16 hares per 100 hectares (247 acres) was estimated (Ebani et al., 2016). Hares were stocked in wood cages and blood samples were drawn from the saphenous vein prior to their release. Animals were checked for the presence of ectoparasites and the age was estimated by palpation of the growing cartilage of the radius and ulna bones, so animals were divided into juvenile hares (<9 months) and adult hares (> 9 months). Nineteen animals were juvenile (8 females and 11 males) while 32 were adult (10 males and 22 females).
Blood samples were collected in sterile tubes with ethylenediaminetetraacetic acid (EDTA) and maintained a 4°C until arrival to laboratory. Diff-Quick stained smears and DNA extraction, with blood/cultured cells genomic DNA extraction mini kit (Trevose, PA, U.S.A.) were performed. DNA samples were stored at 4°C until used in PCR assays.
2.2 Molecular analysis
All DNA samples were submitted to different PCR assays to detect A. phagocytophilum, Bartonella sp., B. burgdorferi sensu lato, C. burnetii, F. tularensis, Leishmania sp. and Babesia sp./Theileria sp., respectively. To detect the first five pathogens the protocols previously described were performed (Relman et al., 1991; Massung et al., 1998; Chang et al., 2000; Milutinovic et al., 2008; Berri et al., 2009).
Piroplasmid DNA was detected following the protocols reported by Cacciò et al. (2002) and Beck et al. (2009). In detail, a nested PCR was performed to amplify a final segment of approximately 560 bp of the 18S rRNA gene.
For the detection of Leishmania DNA, ITS1 gene was chosen as PCR target, as described previously (El Tai et al., 2001); this gene was also used for the discrimination of the Leishmania species involved. Table 1 reports target genes, primers sequences and PCR conditions for each protocol.
PCR amplifications were performed in 50 l of reaction mixtures containing 200 M of deoxynucleoside triphosphates, 0.5 M of each primer, 1.25 U of Taq polymerase (Lucigen Corporation, Middleton, Wisconsin, USA) and 2 l of extracted DNA. All amplifications were
5 performed in the automated thermal cycler Gene-Amp PCR System 2700 (Perkin Elmer, Norwalk, Connecticut, USA).
PCR products were analysed by electrophoresis on 1.5% agarose gel at 100V for 45 min; gel was stained with ethidium bromide and observed. SharpMassTM 100 Plus Ladder (Euroclone, Milano, Italy) was used as DNA marker.
Positive samples were then sequenced on both strands by commercial laboratory BMR- Genomics (Padova, Italy), using the inner primers. The sequence obtained was assembled and corrected by visual analysis of the electropherogram using Bioedit v.7.0.233 and compared with those available in GenBank ® 4 using the BLASTn ® program (http://www.ncbi.nlm.nih.gov/BLAST).
For piroplasmid DNA, the sequence obtained was aligned with 190 sequences described by Lack et al. (2012) using Clustal W (Thompson et al., 1994) and manually trimmed. Then a Bayesian phylogenetic tree was generated in Mr Bayes version 3.1 (Ronquist and Huelsenbeck, 2003) using a general time reversible model including invariable sites (GTR +I). The Bayesian phylogeny was obtained using Monte Carlo Markov chains, which sample every 100 generations over one million generations. Of these trees, 25% were discarded as burn-in material. The remaining trees were used to construct a majority consensus tree using the software FigTree.
3. Results
All the animals appeared in a good health status and nNo ectoparasites were found on examined haresthem.
All hares resulted negative for A. phagocytophilum, Bartonella sp., B. burgdorferi s.l., C. burnetii and F. tularensis.
No haemoparasites were observed in blood smears on 51 hares examined. One sample (1.9%) showed piroplasmid DNA. The sequence obtained, deposited with accession number MF356682, appeared most closely related (showed 97% identity) to with a Babesia isolate obtained from a swine in Sardinia (accession number HQ437690). The phylogenetic analysis collocated this isolate in the clade I (Lack et al., 2012), comprehending most species of Babesia "sensu stricto", asis
shown in Figure 1.
Five hares (9.8%) were positive for Leishmania spp. DNA and sequence analysis revealed in all cases 10099% homology (401/401 bp) with L. infantum, as well as with Leishmania donovani complex, reported in Greece (Tsokana et al., 2016)in all cases.
4. Discussion
The present transversal study was carried out on blood as unique biological specimen since
6 investigate animals were kept in cages, then released after sampling.
XxxxxxAll the animals were not harboring ectoparasites when captured, even if in studies previously carried out on hares living in the same area in past years very few ticks identified as Ixodes ricinus were recovered (data not shown). From a general point of view hares would seem not to be prone to ectoparasites, except for subjects investigated by Psaroulaki et al, (2014a) and Orkun and Karaer (2017) who reported heavy infection by ticks (Rhipicephalus spp. and Ixodes ventalloi and Hyalomma spp., Haemaphysalis parva, Rhipicephalus turanicus, respectively). Other reports deal with fleas (Psaroulaki et al., 2014b), Anoplura such as Hemodipsus lyriocephalus (Girard, 1950) and ticks as I. ricinus (Girard, 1950; Astobiza et al., 2011; Tomaso et al., 2017)
zecche non trovate discorso su ectoparassiti temperature average. Eventuale impatto non a gennaioHowever, the sampling time (winter) is not very favorable to occurrence of ticks on the host.
The present transversal preliminary study was carried out on blood as unique biological specimen since investigate animals were kept in cages, then released after sampling, for these reasons it was not possible to have other samples to perform analysis on further genes to carry out phylogenetic investigation.
All the tested hares resulted negative for the investigated bacterial pathogens. The negative results for F. tularensis, compared with those obtained in previous studies testing other wild animal species, suggest that currently the agent is not present in this geographic area.
In the past, F. tularensis was known as causative agent of human outbreaks in northern and central Italy (Greco et al., 1987; Fabbi et al., 2009), but epidemiological data about the spreading of this pathogen among wildlife in that period are not available. A recent investigation among wild rodents did not find F. tularensis infection cases (Pascucci et al., 2015). Even though F. tularensis currently seems to be not present in Italy, it has been frequently reported in European countries. In fact, molecular investigations have found infected humans and wild animals, including hares, in Germany (Müller et al., 2013), France (Moinet et al., 2016), the Netherlands (Rijks et al., 2013), Norway (Nordstoga et al., 2014), Hungary (Kreizinger et al., 2013), Czech Republic (Hubálek and Rudolf, 2017), Poland (Moniuszko et al., 2011), Spain (Ariza-Miguel et al., 2014), Denmark (Byström et al., 2005), Switzerland (Origgi et al., 2014).
Information about bartonellosis in lagomorphs come from two previous investigations that found B. alsatica in European wild rabbits (Oryctolagus cuniculus) in Spain (Márquez, 2010; Márquez, 2015), whereas a study carried out in California did not detect bartonellae in blood specimens from riparian brush rabbits (Sylvilagus bachmani riparius) (Schmitz et al., 2014).
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7 Considering the spreading of Bartonella species in wild environments, including the Italian ones (Sanogo et al., 2003, Ebani et al., 2015), it could be supposed that hares are animals not very sensitive to these bacteria.
Wild hares seem to have no relevant role in the epidemiology of A. phagocytophilum. Our negative results are in agreement with previous studies that did not found positive hares for this agent (Polin et al., 2004; Yang et al., 2017)
Similarly, C. burnetii infection seems to be rare among hare population. Astobiza et al. (2011) found 9.1% of PCR positive hares in an endemic area of Spain, and Psaroulaki et al. (2014a) found the 48% in Cyprus, but other reports about coxiellosis in lagomorphs are not present in literature. Our negative results could be related to a low prevalence of the pathogen in wildlife and/or a hares’ low sensitivity.
Even though wild hares are considered reservoir hosts for B. burgdorferi s.l. in Europe (Lindgren and Jaenson, 2006), it seems that spirochaetal infections rarely occur in these animals. Also for what concerns this pathogen in lagomorphs, only few data are available in literature. Kjelland et al. (2011) found B. afzelii and B. burgdorferi sensu stricto in hares’ skin samples in Norway and serological surveys detected prevalence values of 41.6% in Czech Republic (Sykora et al., 1990) and 65.8% in Italy (Ebani et al., 2016).
There are few reports of piroplasms in hares and the parasites were mostly morphologically described. The first report of a piroplasm species from hares is due to Dschunkowsky and Luhs (1909) who described a polymorph piroplasm named Piroplasma leporis polymorphe. Later Babesia leporina (Baldelli, 1960) and Theileria leporis (Vaccari et al., 1961) were described in Italy in dead free ranging L. europaeus. Babesia leporis was originally described in hares (L. europaeus) (Levine, 1971; Ristic and Levis, 1977) and in 1995 Horak et al. referred the occurrence of this parasite species in blood smears from scrub hares (L. saxatilis) in South Africa. The unique molecular characterization of Babesia from hare was recently reported from Turkey (Orkun and Karaer, 2017). Therefore, at the best of our knowledge, this is the first molecular report of piroplasms occurring in wild hares from Italy, and the second worldwide.
Molecular analysis appears to be very useful in defining the taxonomy of these parasites, since classical taxonomy criteria (morphology, host range) hold limited value (Lack et al., 2012). Sequences of 18S rRNA gene were used to study the phylogeny of these parasites. Lack et al (2012) and Schnittger et al. (2012) in fact, although reporting not completely agreeing results, converge upon one single clade for Babesia "microti group". Phylogenetic analysis strongly suggests the attribution of the parasite described in this study to clade I.
Comparison with piroplasms isolated from hares available from literature, seems to indicate that
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8 Babesia found in the present study would be closely related to 1 of the 2 isolates reported from Turkey (MF040152), differing from it for 1 single nucleotide. Additional phylogenetic analysis based on other genes would be welcome to an epidemiological investigation ((Paparini et al., 2014; Paparini et al., 2015; Aktas and Ozubek, 2017).
Five hares harbored L. infantumLeishmania DNA in blood, with a sequence similarity of 99% with both L. infantum and L. donovani complex from hares in Greece (Tsokana et al., 2016). The prevalence obtained in the present survey (9.8%) was lower than 43.6% reported from spleen of hares in Spain (Ruiz-Fons et al., 2013). However, blood is considered a less sensitive tissue specimen, so the real occurrence of Leishmania DNA would be underestimated. Dogs are the main reservoirs for these parasites and canine leishmaniasis is endemic in Italy, even though it has been also described in cats, equines, and wild mammals including lagomorphs. Furthermore, both L. granatensis and L. europaeus were proven to harbor parasite DNA in spleen, suggesting that hares may play an unexpected role in the epidemiology of L. infantumLeishmania in Spain (Ruiz-Fons et al., 2013) and Greece (Tsokana et al., 20165). Even though clinical leishmaniosis has never been reported in hares, these animals are considered able to act as reservoirs for both human and dogs in endemic foci. In a previous serologic survey carried out on free ranging hares living in the same area, low antibody titers were reported (Ebani et al., 2016), so suggesting a circulation of the parasite.
5. Conclusion
The obtained results would suggest a limited role of wild hares in the epidemiology of the investigated bacterial pathogens.,A. phagocytophilum, as suggested by a previous serological investigation, too (Ebani et al., 2016). Negative results for F. tularensis and C. burnetii may be related to the absence/low presence of the pathogens in the environment where hares livedwere living. For what concerns B. burgdorferi, Iit could be supposed that the obtained negative results can be due to a short bacteraemic phase. would occur in hares, so better results could be obtained analyzing tissue specimens other from blood.
Twhile these animals would seem to contribute have an active role in maintaining the cycle of examined parasites, considered that among hares sharing the same environment, Leishmania seropositive subjects have been previously detected (Ebani et al., 2016). The occurrence of a piroplasm DNA appears of interest; further research is needed in hares from this area. would contribute to a more comprehensive knowledge. Furthermore, the examined hares appeared to be in
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9 good health status, corroborating the hypothesis of a chronic carrier state of some vector borne agents for this animal species.
It could be supposed that a short bacteraemic phase would occur in hares, so better results could be obtained analyzing tissue specimens other from blood. Siccome sono vivi la quantità di sangue che si preleva è limitata quindi per la sierologia abbiamo tenuto conto dei dati presenti in letteratura da lepri viventi nella stessa zona. Si tratta di lavoro preliminare e ulteriori indagini saranno svolte su further hares.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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ha formattato: Tipo di carattere: Times New Roman
ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Corpo (Calibri), 11 pt ha formattato: Tipo di carattere: Non Grassetto ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano
13 Nordstoga, A., Handeland, K., Johansen, T.B., Iversen, L., Gavier-Widén, D., Mattsson, R., Wik-Larssen, K., Afset, J.E., Næverdal, R., Lund, A., 2014. Tularaemia in Norwegian dogs. Vet. Microbiol. 173, 318-322.
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ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) +Titoli (Cambria), 13 pt, Grassetto, Colore carattere: Evidenziatore 1, Italiano (Italia) ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano ha formattato: Car. predefinito paragrafo, Tipo di carattere: (Predefinito) Times, 24 pt, Grassetto, Italiano Formattato: Giustificato, Interlinea: 1.5 righe Formattato: Interlinea: 1.5 righe
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