Molecular Evidence of Rickettsia slovaca in Wild Boar Lice,in Northeastern Algeria

Introduction

R ickettsia slovaca, a spotted fever group rickettsia, was isolated in 1968 from Dermacentor marginatus ticks in the former Czechoslovakia (Parola et al. 2013). The first case of human infection with R. slovaca was documented in 1997 and was then recognized as a human pathogen (Raoult et al. 1997). R. slovaca has been associated with a syndrome characterized by scalp eschars and neck lymphadenopathy following tick bites. This syndrome was named TIBOLA (tick-borne lymphadenopathy) or DEBONEL (Dermacentor-borne necrotic erythema and lymphadenopathy) (Parola et al. 2013). The term “SENLAT” (for scalp eschar associated with neck lymphadenopathy after a tick bite) has also been recently proposed (Parola et al. 2013). Since 1997, human infections with R. slovaca have been reported regularly in Europe, mainly in Spain, Hungary, Slovakia, Bulgaria, Italy, Germany, and Portugal (de Sousa et al. 2013, Parola et al. 2013).

Dermacentor reticulatus and D. marginatus, commonly known as the ornate dog tick and the ornate sheep tick, respectively, are both known as the main vectors of R. slovaca. These two tick species are known to be ditropic: adults infest large mammals such as sheep, cattle, goats, and wild boars, while larvae and nymphs feed mostly on small mammals and medium-sized carnivores. D. reticulatus is most frequently found in colder northern areas of western Europe and the former Soviet Union, with high humidity and mild winters. D. reticulatus is primarily dog and carnivore tick, but it can be found on ungulates such as sheep, cattle, and horses (Parola et al. 2009). The D. marginatus tick is recognized as the main vector and reservoir for R. Slovaca in Mediterranean areas, including southern Europe, where dense bush and tree cover are common, as well as under oak and pine vegetation. D. marginatus is also prevalent in North Africa, in the cooler and more humid areas associated with the Atlas Mountains. D. marginatus and D. reticulatus have been suggested as reservoirs of R. slovaca, which occur in ticks through transstadial and transovarian transmission. The geographic distribution of these rickettsiae, therefore, is likely to parallel that of the Dermacentor tick. A high prevalence of ticks infected with this bacterium has been found in a great majority of European countries (Parola et al. 2013).

In Algeria, R. slovaca was detected in D. marginatus ticks collected from vegetation in the Blida region in 2012 (Kernif et al. 2012) and in the Souk Ahras regions (Leulmi et al. 2016). In addition to ticks, wild boars are regularly parasitized by lice, including Haematopinus suis. H. suis is the only species of louse found on pigs and boars (Wall and Shearer 2001). It prefers the fold of the neck and jowl, around the ears, and on the flanks and back of pigs and boars. Since, both H. suis and D. marginatus feed on the same animal than recognized vectors of R. slovaca, our study aims to assess the presence and identity of Rickettsiae in lice collected from wild boars and to evaluate the relationship that may exist between R. slovaca and H. suis. Overall, the possible presence of R. slovaca in lice could expand the knowledge about the repertoire of vector-lice diseases in Africa and mainly in Algeria and to define whether boar's lice play a role in the epidemiology of R. slovaca.

Materials and Methods

Between April 2011 and April 2016, an investigation in the far northeast of Algeria (Annaba and El Tarf) was conducted on the presence of Rickettsia spp. in boar lice. In the common mountains between Annaba and El Tarf, the boars were flushed out with the help of approved hunting associations and, once captured, were inspected for the presence of lice. Lice were harvested directly on the boar using clamps. Once recovered from their hosts, lice were identified at the genus and species levels, using available keys identification (Wall and Shearer 2001). Before their genomic DNA extraction, lice were rinsed twice in distilled water for 10 min and dried on sterile filter paper.

A total of 100 μL of DNA was extracted from one half of each louse using the QIAamp Tissue Kit (QIAGEN, Hilden, Germany) and the BioRobot EZ1 (QIAGEN), according to the manufacturer's instructions. DNA was used as template for quantitative real-time polymerase chain reaction (qPCR). We used the RKND03 system, targeting the long fragment of the gltA gene of Rickettsia spp. (Socolovschi et al. 2010). qPCRs were performed by using the CFX96 Real-Time System C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Singapore). Positive results were confirmed using a standard PCR targeting the OmpA gene of Rickettsia spp. (Socolovschi et al. 2010). Negative controls were used in each PCR (qPCR and standard PCR) and consisted of DNA extracted from Rickettsia-free R. sanguineus ticks from our laboratory colony, while DNA from a R. montanensis strain cultured in our laboratory was used as a positive control.

The success of the PCR amplification was verified by electrophoresis migration in 2% agarose gel. Products were purified by using a NucleoFast 96 PCR plate (Macherey-Nagel EURL, France) as recommended by the manufacturer. Purified PCR products were sequenced using the OmpA primers and the BigDye version 1–1 Cycle Ready Reaction Sequencing Mixture (Applied Biosystems, Foster City, CA) in the ABI 31000 automated sequencer (Applied Biosystems). Sequences were then assembled and analyzed using ChromasPro version 1.34 software (Technelysium Pty Ltd., Tewantin, Queensland, Australia), then the sequences of Rickettsia species amplified in the present study was compared with other OmpA sequences of Rickettsia species available on GenBank database (Fig. 1).

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FIG. 1.

Phylogenetic tree demonstrates the position of Rickettsia slovaca of the present study with other Rickettsiae.

Results

From the 117 boars captured, 325 lice were recovered and identified as Haematopinus suis. Twenty-one boars out of 117 (17.9%) were found infested with lice, where between 8 and 21 lice were harvested by boar, making an infestation average of 15.5 lice/infested boar. A total of 92 lice were randomly selected to be screened for the detection of rickettsiae. Overall, 7.61% (7/92) were positive for Rickettsia spp. using qPCR RKND03 system. The positive lice were removed from five different boars (one louse positive by boar for four boars and a last boar with three lice collected from the same boar). Using standard PCR targeting the OmpA gene, R. slovaca (100% similarity with the R. slovaca strain WB2/Dm Pavullo outer membrane protein A gene, partial cds (760/760 bp, GenBank accession no. HM161787.1) was identified.

Discussion

The detection of DNA R. slovaca in Haematopinus suis does not currently signify that they are a vector. Indeed, lice could acquire the bacteria after feeding on a bacteremic boar. In a recent study in Spain, the seroprevalences of R. slovaca in domestic ruminants were reported to be 16% in sheep, 21% in goats, and 65% in bull fighting cattle. In addition, R. slovaca DNA was detected in a goat blood sample (Ortuño et al. 2012), suggesting that R. slovaca may be circulating in domestic ruminants, mainly hairy domestic and wild animal hosts of D. marginatus. Melophagus ovinus flies around the Taklimakan Desert located in China tested positive for R. slovaca DNA (Liu et al. 2016), which suggests possible transmission through arthropods other than Dermacentor ticks. In Algeria, this is the first report of the presence of R. slovaca in lice infesting boars and the second report of the presence of R. slovaca in vectors infesting boars.

Given that the clinical manifestations of R. slovaca are zoonotic diseases and to determine whether wild boar's lice are a vector of R. slovaca, epidemiological studies need to be conducted. However, the proven vector of R. slovaca, (D. marginatus) is overabundant in Algeria, Maghreb, and in the northern areas of Africa (Leulmi et al. 2016). The presence of R. slovaca DNA on H. suis would enable to determine the relationship between R. slovaca and lice; more specifically, H. suis. Despite the fact that several investigations have suggested that wild boar may have a potential role in the eco-epidemiology of R. slovaca (Ortuño et al. 2007), the role of boars as reservoirs, amplifiers, or simply hosts of the R. slovaca vector is also yet to be identified. Based on the present investigation and other studies, we suggested, the possible presence of an epidemiological network implicated in the circulation, transmission, and the maintain of R. slovaca in wild animals involving the wild boars, D. marginatus ticks, and the boar's lice H. suis.