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Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria
Comparative Immunology, Microbiology and Infectious Diseases 50 (2017) 34–39
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Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria Atef Aouadi a,b , Hamza Leulmi c,d , Mehdi Boucheikhchoukh a , Ahmed Benakhla a , Didier Raoult c , Philippe Parola c,∗ a
Université Chadli Bendjdid, Département des Sciences Vétérinaires, El Tarf, 36000, Algeria Université Mohamed Cherif Messaadia, Institut des Sciences Agronomiques et Vétérinaires, Souk Ahras, 41000, Algeria c Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar), Inserm 1095, Marseille, France d Ecole Nationale Supérieure Vétérinaire d’Alger. El Aliya Alger, 16000, Algeria b
a r t i c l e
i n f o
Article history: Received 9 August 2016 Received in revised form 26 October 2016 Accepted 13 November 2016 Keywords: Theileria ovis Babesia ovis Small ruminants Ticks Algeria
a b s t r a c t Using qPCR, standard PCR and/or sequencing, we investigated the presence of tick-associated microorganisms in ticks and blood from sheep and goats from Souk Ahras, Algeria. Borrelia theileri, was detected in (7/120, 5.8%) blood from sheep and (13/120, 10.8%) goats. Anaplasma ovis was screened in (38/73, 52%) Rhipicephalus bursa and (5/22, 22.7%) R. turanicus and in (74/120, 61.7%), (65/120, 54.2%) blood of sheep and goats respectively. Coxiella burnetii tested positive in R. bursa (4/73, 5.5%) and (7/120, 5.8%) blood of sheep and (2/120, 1.7%) goats. Theileria ovis was detected in (50/147, 34%) R. bursa and (3/22, 13.6%) R. turanicus and in (64/120, 53.3%) blood of sheep and (25/120, 20.8%) goats. Babesia ovis was screened positive in (23/147, 15.6%) R. bursa and (7/48, 14.6%) R. turanicus. Our findings expand knowledge about the repertoire of tick-borne microorganisms present in ectoparasites and/or the blood of small ruminants in Algeria. © 2016 Published by Elsevier Ltd.
1. Introduction Small ruminants are an important source of meat and milk in Algeria and play a vital role in food security. Sheep predominate, at around 80% of total livestock. They are amongst the major economically important livestock in Algeria with about 27 million head in 2014 [1,2]. Goat populations also play an important role in the livelihood of resource-poor farmers and represent around 4 million head [2]. Sheep and goats are exposed to several health problems and their productivity is thought to be greatly reduced due to infectious diseases and parasitic diseases [2–5]. Vector-borne diseases represent a large proportion of these infectious diseases, which are caused by a multitude of pathogens transmitted by arthropod vectors [6]. Ticks were the first arthropods to be established as vectors of pathogens [7] that can transmit
∗ Corresponding author at: Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses Tropicales et Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, Faculté de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France. E-mail address: [email protected] (P. Parola). http://dx.doi.org/10.1016/j.cimid.2016.11.008 0147-9571/© 2016 Published by Elsevier Ltd.
them to humans [8]. They are considered to be the second biggest vectors of pathogens after mosquitoes [9] and the most important vectors of disease-causing pathogens in animals [10]. There are about 900 species of ticks described so far [11], most of which are responsible for the transmission of a huge variety of microorganisms, including viruses, bacteria and parasites [12]. Theileria spp. and Babesia spp., the causative agents of theileriosis and babesiosis, respectively, are among the most economically important hemoparasitic tick-borne diseases in ruminants worldwide [13]. In the past, most attention has been given to bovine-infecting piroplasmosis, and most tick-borne diseases in small ruminants have received less consideration [14]. However, because of the socio-economic impact in a number of countries, interest has recently increased in sheep and goat piroplasmosis [14]. The Theileria species infecting small ruminants are Theileria ovis, T. lestoquardi, T. luwenshuni, T. uilenbergi, T. recondita and T. separata [15,16]. Small ruminants are also affected by Babesia ovis, B. motasi, B. crassa, B. foliata, B. taylori, Babesia sp. (China) and Babesia sp. (Xinjiang) [17]. Likewise, a vast number of tick-related bacteria have been identified in the past 25 years [18], including borrelioses [19],
10 (5 b + 5 t) 10 (5 b + 5 t) 10 (5 b + 5 t) 00 10 (10 b) 00 10 (7 b + 3 t) 10 (10 b) 10 (8 b + 2 t) 10 (10 b) 10 (8 b + 2 t) 05 (5 b)
From goats From sheep R. turanicus R. bursa
35 (28 f + 7 m) 3 (1 f + 2 m) 54 (48 f + 6 m) 00 10 (8 f + 2 m) 2 (1 f + 1 m) 18 (3 f + 15 m) 00 19 (14 f + 5 m) 00 18 (14 f + 4 m) 10 (5 f + 5 m) 68 (33 f + 35 m) 4 (3 f + 1 m) 00 00 00 00 28 (3 f + 25 m) 2 (2 f) 00 00 12 (3 f + 9 m) 00 283 6/10 7/10 4/10 3/10 2/10 3/10 6/10 00/10 00/10 4/10 00/10 3/10 f; female, m; male, b; Rhipicephalus bursa, t; Rhipicephalus turanicus
R. turanicus R. bursa
75 (55 f + 20 m) 29 (11 f + 18 m) 95 (72f + 23 m) 16 (9 f + 7 m) 129 (110 f + 19 m) 30 (8 f + 22 m) 00 00 87 (80 f + 7 m) 00 00 00 82 (80 f + 2 m) 3 (3 f) 35 (20 f + 15 m) 00 10 (8 f + 2 m) 2 (2 m) 17 (5 f + 12 m) 00 10 (8 f + 2 m) 2 (2 m) 5 (5 f) 00 627 104 111 159 00 87 00 85 35 12 17 12 05 627 10/10 10/10 9/10 00/10 5/10 00/10 6/10 3/10 4/10 2/10 3/10 1/10
Ticks species and sex Total of tick collected from sheep Infested sheep /total of sampled sheep Sites of the study
Table 1 Ticks collection and blood sampling.
This cross-sectional study was carried out on ticks and blood from sheep and goats on 12 sites (Table 1) through the province of Souk Ahras in the Northeastern of Algeria, (36◦ 17 15 N 7◦ 57 15 E). Souk Ahras is surrounded by wooded mountains and is 1000 m above sea level. It is an extension of the Telli Atlas mountains. It has a semi-humid climate characterized by a hot summer and a cold, wet winter with rainfall averaging 850 mm per year, the bulk of which is seen between October and April. Small ruminants in all farms in the study were kept under a semi-extensive system, characterized by free grazing on pastures. Sheep and goats were kept under semi-enclosed systems at night and allowed to graze on pastures where the animals mixed with cattle, dogs and horses grazing in the same area. Twelve farms throughout the province of Souk Ahras were included in this study according to the cooperation degree of their owners. Ten sheep and 10 goats were randomly selected and sampled per farm. A total of 120 Barbarine sheep blood and 120 Arbi goat blood samples were collected individually in EDTA Vacutainer tubes from small ruminants. This cross-sectional survey was done in April 2014 and June 2015, periods when ruminants graze in pastures and are exposed to ticks and tick-borne diseases. With oral permission from all of the animals’ owners, blood samples were taken from apparently healthy sheep and goats and collected by cephalic venipuncture into tubes. The blood samples were collected from randomly selected male and female small ruminants including adults and yearlings. Once the blood was sampled, the entire body of each of the 240 animals was inspected for ticks. In order to reduce the time of restraint and handling of the animal we focused particularly on the ears, neck, udders and external genitalia (predilection sites of the ticks attachment). Ticks were removed manually and using blunted clockmakers’ forceps and immediately placed in 70% ethanol inside tubes labeled with the identification number and the date of collection. Only a portion of the collected ticks was used for the present study. All biological materials including blood and ticks were forwarded to Marseille, France, for morphological identification of ticks at the species level using morphological criteria within standard taxonomic keys [30], and stored at −20 ◦ C prior to molecular analyses (both blood and ticks).
Infested goats/ total of sampled goats
2.1. Study areas, tick collection and blood sampling
Ouilen Sedrata Khedara Mechrouha Merahna Hedadda Souk Ahras Medaourouch Oum Adayem Oued Kabarite Taraguelte Henancha Total
2. Materials and methods
38 54 12 18 19 28 72 00 00 30 00 12 283
Total of tick collected from goats
Ticks species and sex
Total tick qPCR tested
anaplasmosis [20], Q fever [21] and tick-borne rickettsial diseases [18]. In recent years, the prevalence of tick-borne bacterial diseases has dramatically increased and emerged throughout the world [22]. Spirochaete Borrelia theileri has been identified in the blood of various domestic mammals including ongulates, mainly cattle and small ruminants in tropical and subtropical regions of Africa, where the genus Boophilus (Rhipicephalus) was recognized as vector [23]. The clinical signs of infection in ruminants and other animals remain mild and variable, but usually include fever and anemia [24]. Anaplasma ovis is an erythrocytic anaplasma that parasitizes red blood cells [25] and may cause mild to severe disease in sheep and other ruminants [26]. A. ovis is transmitted primarily by Rhipicephalus bursa ticks [27]. In addition, Coxiella burnetii, the causative agent of Q fever, is recognized as a worldwide highly infectious zoonotic intracellular bacterium [28]. C. burnetii is able to infect various species of domestic mammals, including small ruminants and humans [29]. In this study, we aimed to update the occurrence and repertoire of tick-borne parasitic and bacterial diseases in Algeria by using molecular-based methods.
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10 (7 b + 3 t) 10 (10 b) 10 (8 b + 2 t) 10 (10 b) 10 (10 b) 10 (5 b + 5 t) 10 (6 b + 4 t) 00 00 10 (8 b + 2 t) 00 10 (10 b) 185
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2.2. DNA extraction Prior to DNA extraction, a convenient sample of ticks was selected according to a good representation of species and hosts, however all blood samples were processed to extract their DNA. All experiments and handling of blood and ticks were conducted in a laminar flow biosafety hood. A total of 200 L of blood was transferred to a specific tube prior to extraction using the QIAamp tissue kit (Qiagen, Hilden, Germany) and EZ1 robot as described by the manufacturer. Each ethanol-conserved tick was rinsed twice in sterile water for 10 min and dried on filter paper. Each sample was then longitudinally incised using an individual scalpel into two parts, one on which was crushed in sterile tubes (Eppendorf; Hamburg, Germany). The remaining portion of each tick was kept at −80 ◦ C for further control. Each tick sample was incubated overnight at 56 ◦ C in 180 L of buffer G2 and 20 L of proteinase K for pre-lysis followed by extraction using QIAamp tissue kit (Qiagen, Hilden, Germany) and EZ1 robot. For all samples (ticks and blood), the final elution volume was 100 L. 2.3. Molecular tests Once DNA had been extracted, it was used in real time PCR template assays to detect bacteria (Borrelia spp., Anaplasma spp., Coxiella burnetii, Bartonella spp. and Rickettsia spp.) and protozoan parasites (Theileria spp. and Babesia spp.). The qPCR reaction mixture consisted of 5 L of DNA and 15 L of mix from the Takyon PCR Kit (Qiagen, Hilden, Germany) as described [3]. Two negative controls were used in each qPCR plate and consisted of 5 L DNA extracted from uninfected ticks from our laboratory colony and 15 L takyon mix. Positive controls included DNA extracted from a positive samples for Theileria spp. and Babesia spp. and dilution of cultured strains of Borrelia crocidurae (for the detection of Borrelia spp.), Anaplasma phagocytophylum (for the detection of Anaplasma spp.), Coxiella burnetii (for the detection of C. burnetii), B. elizabethae (for the detection of Bartonella spp.) and R. montanensis (for the detection of Rickettsia spp.). Results were deemed positive if the cycle threshold value obtained by CFX96TM was lower than 35. All positive results were confirmed with a second qPCR system and/or sequence reaction. 2.3.1. Detection of Borrelia spp All DNA samples (ticks and blood) were individually tested for the presence of Borrelia spp. using the Bor16S system, which targets the rrs gene that encodes the 16S subunit of Borrelia genus-specific as described elsewhere [31]. Samples positive for Borrelia spp. by qPCR were further subjected to standard PCR to amplify a portion of the flaB (flagellin) and 16S rRNA genes of Borrelia spp. as described previously [31]. The success of the PCR amplification was verified by 1.5% of Agarose gel migration, with ethidium bromide at 130 V for 25 min. The bands were visualized under ultraviolet illumination using BenchTop pGEM® DNA Marker (Promega, Madison, Wisconsin, USA) to estimate the size of the products. The PCR products were then purified using the NucleoFast 96 PCR plate MacheryNagel EURL, France, as recommended by the manufacturer. The obtained amplicon was sequenced in the ABI 31000 automated sequencer (Applied Biosystems) by using the DNA sequencing BigDye Terminator Kit (Perkin- Elmer, Waltham, MA) as described by the manufacturer. The sequences obtained were assembled using ChromasPro 1.7 (Technelysium Pty Ltd., Tewantin, Australia) and compared with borrelias sequences in the GenBank database using BLAST: (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE TYPE=BlastSearch&LINK LOC=blasthome).
2.3.2. Detection of Anaplasma spp. All samples were primarily screened by qPCR to determine the presence of Anaplasmataceae in the DNA from tick and blood samples. Primers and a Taqman® probe targeting the 23S rRNA gene were used to amplify an amplicon of 280 bp as described elsewhere [32]. Positive results were confirmed by standard PCR and sequenced as described in the Detection of Borrelia spp. section using two systems of standard PCR targeting 16S Ehr and 23S [32]. 2.3.3. Detection of Coxiella burnetii As a first step, C. burnetii bacterial DNA was detected by qPCR system with C. burnetii–specific primers and a probe designed to amplify the IS1111 gene spacer [33]. Next another qPCR that amplified the IS30a spacer was used to confirm C. burnetii–positive results [3]. 2.3.4. Detection of Rickettsia spp. All the extracted DNA was assessed for the presence of rickettsial DNA by using a Rickettsia genus-specific qPCR for spotted-fever group Rickettsia by targeting the gltA gene (RKND03 system) [34]. 2.3.5. Detection of Bartonella spp. All DNA samples from ticks and blood were individually tested for the presence of the 16S–23S ribosomal RNA intergenic spacer gene by qPCR with a 21-base pair probe specific for the genus Bartonella as described [3]. 2.3.6. Detection of Theileria spp. and Babesia spp. Initially all samples were screened by qPCR targeting a fragment of the 28S to detect Theileria spp. using the following set of primers (Thei 28s F: 5 -NTACAGACTCAAAGGATCGATAGGC-3 and Thei 28s R: 5 - AAGGCGAACTCAATGAGGACA-3 ) and the probe (Thei 28s P: 6FAM- TCAAGCGAACTTTTCCCCTTTTGGTC-TAMRA). The presence of Babesia spp. DNA in ticks and blood samples was also determined by qPCR targeting 18S using the following primers (Bab 18s F: 5 -TTGGGGGCATTCGTANTNRAC-3 and Bab 18s R: 5 -TTCTTGATTAATGAAAACGTCTTG-3 ) and the probe (Bab 18s P: 6FAM-AAGACGAACTACTGCGAAAGCATTTGC-TAMRA). We designed both systems to detect Theileria spp. and Babesia spp. in our laboratory. Positive results were confirmed and sequenced by standard PCR targeting 18S rRNA. The 18S ribosomal RNA gene was amplified by PCR, with a pair of generic apicomplexan 18S rRNA-specific primers: CRYPTOF, the forward primer (5 AACCTGGTTGATCCTGCCAGT-3 ), and CRYPTOR, the reverse primer (5 GCTTGATCCTTCTGCAGGTTCACCTAC-3 ) as described elsewhere [35]. The conditions for the standard PCR included 95 ◦ C for 15 min, followed by 39 cycles of denaturation at 94 ◦ C for 30 s, annealing at 65 ◦ C for 30 s, and extension at 72 ◦ C for 1.5 min. The final extension was done at 72 ◦ C for 9 min, followed by a hold step at 15 ◦ C [36]. 3. Results 3.1. Tick identification and sample collection A total of 910 ticks were collected from sheep (627/910, 68.9%) and goats (283/910, 31.1%) from the province of Souk Ahras. Two tick species were identified among overall collected ticks: Rhipicephalus bursa (545/627, 86.9%) and Rhipicephalus turanicus (82/627, 13.1%), collected from 120 sheep and R. bursa (262/283, 92.6%) and R. turanicus (21/283, 7.4%), collected from 120 goats. From all the ticks collected, a convenient sample of 95 ticks sampled on sheep (73 R. bursa and 22 R. turanicus) and 90 ticks collected on goats (74 R. bursa and 16 R. turanicus) were PCR processed in this
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Table 2 Detection of ticks borne micro-organisms in blood and ticks from sheep, goats. Sites of the study Ouilen Sedrata Khedara Mechrouha Merahna Hedadda Souk Ahras Medaourouch Oum Adayem Oued Kabarite Taraguelte Henancha Total
Borrelia theileri
Anaplasma ovis
Coxiella burnetii
Theileria ovis
Babesia ovis
– 2 s, 5 g – 5g 3 s, 2 g – – – – – 1g 4s 7 s, 13 g
8 s, 8 g, 1 b 6 s, 5 g, 2 b, 2 t 5 s, 2 g, 3 b, 3 t 6 s, 9 g 6 s, 5 g, 7 b 3 s, 5 g 7 s, 1 g, 5 b 6 s, 8 g, 9 s, 2 g, 7 b 8 s, 9 g, 8 b 4 s, 6 g, 3 b 6 s, 5 g, 2 b 74 s, 65 g, 38 b, 5 t
– 2s 3 s, 4 b – 2s 2g – – – – – – –
8 s, 4 g, 3 b 2 s, 4 b 5 s, 6 g, 3 t, 2 Rb 3 s, 2 g, 2 Rb 5 s, 4 g, 7 b 5 s, 2 g 1 s, 6 b, 1 Rb 5 s, 4 g, 6 b 9 s, 6 s 7 s, 6 b, 2 Rb 6 s, 7 b 8 s, 3 g, 4 b 64 s, 43 b,
1 t, 5 Rb – 4 b, 2 t, 2 Rt – – – 5 Rb – – 7b – 2 b, 2 t –
b; Rhipicephalus bursa collected from sheep, Rb; Rhipicephalus bursa collected from goats, t; Rhipicephalus turanicus collected from sheep, Rt; Rhipicephalus turanicus collected from goats, s; blood of sheep, g; blood of goats, -; negative
study, with 240 blood samples from 120 sheep and 120 goats, to detect bacteria and protozoan parasites.
ples showed that the closest sequences available in GenBank were those for Theileria ovis: accession nos. FJ603460.1, AY508457.1, AY508456.1, AY260172.1 and EU622911.1.
3.2. Detection of bacteria 3.2.1. Detection of Borrelia spp. Of the 185 qPCR tested ticks, no DNA of Borrelia spp. was detected, however (7/120, 5.8%) sheep blood and (13/120, 10.8%) goat blood were positive using the 16S qPCR system from the Borrelia genus (Table 2). DNA sequence analyses of the PCR products targeting the 16S and flab on the Borrelia blood positive showed 100% similarity with Borrelia theileri (GenBank accession no. KF569941.1, 1206/1206 bp), regardless of the origin. 3.2.2. Detection of Anaplasma spp. Overall 23S qPCR tested ticks, (38/73, 52%) R. bursa and (5/22, 22.7%) R. turanicus collected on sheep were positive (Table 2). Anaplasma spp. was also screened positive in sheep blood (74/120, 61.7%) and goat blood (65/120, 54.2%). Sequencing analysis using the 16S Ehr system was performed on Anaplasma spp. positive ticks (10 samples) and blood (10 samples) and was not discriminating among Anaplasma ovis, A. marginale, and A. centrale, showing 100% similarity with these three Anaplasma species. The sequencing results using the 23S standard PCR system showed that all sequences of Anaplasma spp. detected in ticks and blood were similar to the sequence of Anaplasma ovis (Anaplasma ovis strain KMND Niayes 14 23S ribosomal RNA gene, partial sequence, GenBank accession no. KM021411.1). 3.2.3. Detection of Coxiella burnetii DNA from C. burnetii was detected in R. bursa (4/73, 5.5%) collected on sheep, blood of sheep (7/120, 5.8%) and blood of goats (2/120, 1.7%) (Table 2). We confirmed the result using a second qPCR system. 3.2.4. Detection of Rickettsia spp. and Bartonella spp. All the selected 185 ticks, 120 sheep and 120 goat blood samples tested negative for Rickettsia spp. and Bartonella spp. 3.3. Detection of haemoprotozoan-parasites 3.3.1. Detection of Theileria spp. Of the 185 tested ticks, 53 samples; (43/73, 58.9%) R. bursa and (3/22, 13.6%) R. turanicus collected on sheep, and (7/74, 9.4%) R. bursa sampled on goats, were positive for genus-specific Theileria by qPCR. Sixty-four sheep and 25 goat blood samples were also positive using the Theileria-qPCR system (Table 2). Subsequent sequencing of the 18S gene amplicons from all positive PCR sam-
3.3.2. Detection of Babesia spp. Overall in ticks and blood qPCR tested, Babesia spp. was screened positive in (13/73, 17.8%) R. bursa and (5/22, 22.7%) R. turanicus collected on sheep, and (10/74, 13.5%) R. bursa and (2/26, 7.7%) R. turanicus sampled on goats (Table 2). No blood was Babesia-qPCR positive. The amplification with sequencing of a fragment of the 18S gene of these Babesia positive samples showed 100% similarity with Babesia ovis accession nos. AY260178.1 and AY150058.1. 4. Discussion This investigation reports the first direct evidence of tick-borne haemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria (Anaplasma ovis, Borrelia theileri and Coxiella burnetii) in ticks (R. bursa and/or R. turanicus) and blood from small ruminants (sheep and goats) in Souk Ahras, Northern Algeria. In our study, the presence of DNA from pathogens in ticks and blood collected from small ruminants was screened using a validated method consisting of double qPCR assays and/or subsequent sequencing methods. The robustness of the data that we report is based on strict laboratory procedures and controls, including rigorous positive and negative controls to validate each test. Vector-borne agents are increasingly recognized as important causes of morbidity and mortality in humans and domestic animals worldwide [37]. In addition, in Algeria, the incidence of tick-borne zoonoses has increased over the last decade [3,31,38]. To the best of our knowledge, this is the first molecular study demonstrating the presence of piroplasms, T. ovis and B. ovis, in ticks and blood from small ruminants in Algeria. Our work on Theileria spp. was carried out on 120 sheep blood samples, 120 goat blood samples and 185 ticks, in which we found 64 (53.3%) sheep, 25 (20.8%) goats and 53 (28.5%) Rhipicephalus ticks that were T. ovis DNA positive. Initially, T. ovis, was considered to be less pathogenic than T. lestoquardi, the agent of malignant ovine theileriosis, which is the major pathogen present in the arid regions of Africa [39]. Until recently, T. ovis was identified mainly by its non-pathogenicity for sheep and goats [14], which could explain the absence of reported clinical theileriosis cases in the studied regions. T. ovis was assessed as positive via reverse-line blotting in R. turanicus ticks and blood collected from sheep and goats from Tunisia [40]. T. ovis from the Mediterranean basin has R. bursa as vector, where it was detected in sheep and goats from Southern Catalonia, Spain [41].
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Babesia ovis was detected in our study, in R. bursa and R. turanicus ticks sampled on sheep and goats. B. ovis, unlike T. ovis, is the causative agent of ovine babesiosis with B. motasi and B. crassa [42]. B. ovis is highly pathogenic to sheep and goats and its pathogenicity is well known [40]. Clinical symptoms of the disease include fever, anemia, loss of appetite, hemoglobinuria, icterus, and death [42]. The R. bursa tick is the principal vector of B. ovis in Mediterranean areas [43] where ovine babesiosis is prevalent, mainly in North Africa [40]. In addition to hemoparasites detected in this investigation, tickborne diseases were also screened positive in our study. We identified the DNA of the spirochaete Borrelia theileri in blood of sheep and goats. B. theileri is the causative agent of bovine borreliosis in cattle [24] and considered to be one of the relapsing fever Borreliae that have been related as being hosted by hard ticks [37]. Rhipicephalus spp. is the main recognized vector in several countries of the world, mainly in tropical and subtropical regions of Africa [23;24]. B. theileri was detected from Boophilus microplus and Rhipicephalus geigyi in Mali [44] and showed 100% similarity with the B. theileri that we found. Generally the clinical signs of infection caused by B. theileri are variable and mild, usually associated with fever and anemia [24]. In this study, Anaplasma ovis was also molecularly identified in the blood of sheep and goats and in R. turanicus and R. bursa ticks sampled on sheep. A. ovis is the causative agent of anaplasmosis in small ruminants, where it invades and propagates within their erythrocytes, as are A. marginale and A. centrale [25]. Clinical signs may be mild to severe [26]. A. ovis is known to be transmitted by R. bursa ticks [27]. In Algeria, there is a lack of information about the Anaplasmataceae infection in animals and mainly in ruminants, where only A. phagocytophilum, A. platys and Anaplasma sp. were found in cattle [32]. Finally, we detected the agent of Q fever, C. burnetii, in R. bursa ticks and blood from sheep and goats. C. burnetii is a highly infectious zoonotic intracellular bacterium [28] that can affect different species of wild and domestic mammals [29]. Q fever has been described worldwide in outbreaks involving sheep, goats, cats, dogs and wild animals, while reservoirs are extensive but only partially known and include mammals, birds, and arthropods, mainly ticks [45]. In Algeria a few human cases of Q fever have been documented, with only two human cases reported in Oran [46]. DNA from C. burnetii was recently reported in ticks from bats in Souk Ahras, Algeria [3] and in dog and cat spleens [47]. Overall our study, we detected Borrelia theileri only in blood of sheep and goats. In opposite, Babesia ovis was screened positive only in ticks of sheep and goats. In addition, Anaplasma ovis, Coxiella burnetii and Theileria ovis DNA were present in ticks and in blood of sheep and goats. In our case, mainly, the positive animals have a positive ticks. In case, if ticks were removed from their hosts, that it could have been bacteremic or parasitemic, the DNA of the detected micro-organism can be present in ticks and in animal blood simultaneously and ticks can be vectors for some pathogens or simple carriers in other cases [3]. However, if the animals is not bacteremic, or parasitemic or the ticks have been infected through the blood of other infected animals and newly attached to a new animal host, in this case ticks can be positive and the animal host not. To date, information about vector-borne diseases agents circulating in Algeria remains limited mainly to hemoparasitic diseases. This paper provides interesting new information on zoonotic pathogens emerging from small ruminants in Northern Algeria. Our findings will help human and veterinary clinicians to enlarge the panel of pathogens to consider in differential diagnosis. Future epidemiological studies using larger numbers of domestic animals and ectoparasites from more localities is necessary in order to determine the actual prevalence and distribution of these diseases in
Algeria. Further investigations are also warranted to isolate these species and determine their clinical significance. Conflict of interests The authors declared that they have no competing interests. Authors’ contributions AA contributed to arthropod and blood collections, performed DNA extractions, qPCRs, sequencing and first drafted the paper. HL helped with the experiments, contributed to the manuscript, analyzed the data, coordinated the study and identified arthropods. AB contributed to conceiving, designing and coordinating the study. DR Contributed reagents/materials/analysis tools and analyzed the data. PP conceived, designed and coordinated experiments. All authors read and approved the final manuscript. Acknowledgements This work was carried out thanks to the support of the A*MIDEX project (no. ANR-11-IDEX-0001-02) funded by the French Government’s “Investissements d’Avenir” program, managed by the French National Research Agency (ANR). References [1] FAO, http://faostat.fao.org. 2016. Ref Type: Online Source. [2] M. Kardjadj, B. Kouidri, D. Metref, P.D. Luka, M.H. Ben-Mahdi, Abortion and various associated risk factors in small ruminants in Algeria, Prev. Vet. Med. 123 (2016) 97–101. [3] H. Leulmi, A. Aouadi, I. Bitam, A. Bessas, A. Benakhla, D. Raoult, et al., Detection of Bartonella tamiae, Coxiella burnetii and rickettsiae in arthropods and tissues from wild and domestic animals in northeastern Algeria, Parasites Vectors 9 (2016) 27. [4] S.E. Merdja, H. Khaled, R. Aaziz, F. Vorimore, C. Bertin, A. Dahmani, et al., Detection and genotyping of Chlamydia species responsible for reproductive disorders in Algerian small ruminants, Trop. Anim. Health Prod. 47 (2) (2015) 437–443. [5] B. Bentounsi, R. Trad, N. Gaous, K. Kohil, J. Cabaret, Gastrointestinal nematode resistance to benzimidazoles on a sheep farm in Algeria, Vet. Rec. 158 (18) (2006) 634–635. [6] G. Baneth, Tick-borne infections of animals and humans: a common ground, Int. J. Parasitol. 44 (9) (2014) 591–596. [7] F. Dantas-Torres, B.B. Chomel, D. Otranto, Ticks and tick-borne diseases: a One Health perspective, Trends Parasitol. 28 (10) (2012) 437–446. [8] L.Q. Fang, K. Liu, X.L. Li, S. Liang, Y. Yang, H.W. Yao, et al., Emerging tick-borne infections in mainland China: an increasing public health threat, Lancet Infect. Dis. 15 (12) (2015) 1467–1479. [9] P. Parola, D. Raoult, Ticks and tickborne bacterial diseases in humans: an emerging infectious threat, Clin. Infect. Dis. 32 (6) (2001) 897–928. [10] O. Mediannikov, F. Fenollar, Looking in ticks for human bacterial pathogens, Microb. Pathog. 77 (2014) 142–148. [11] A.A. Guglielmone, R.G. Robbins, D.A. Apanaskevich, T.N. Petney, A. Estrada-Pena, I.G. Horak, The Hard Ticks of the World (Acari: Ixodida: Ixodidae), 2014. [12] E. Tijsse-Klasen, M.P. Koopmans, H. Sprong, Tick-borne pathogen − reversed and conventional discovery of disease, Front. Public Health 2 (2014) 73. [13] G. Uilenberg, International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health, Vet. Parasitol. 57 (1-3) (1995) 19–41. [14] H. Yin, L. Schnittger, J. Luo, U. Seitzer, J.S. Ahmed, Ovine theileriosis in China: a new look at an old story, Parasitol. Res. 101 (Suppl. 2) (2007) S191–S195. [15] M.A. Darghouth, Piroplasmids of livestock in Tunisia, Arch. Inst. Pasteur Tunis 81 (1-4) (2004) 21–25. [16] A.H. El Imam, S.M. Hassan, A.A. Gameel, A.M. El Hussein, K.M. Taha, M.C. Oosthuizen, Molecular identification of different Theileria and Babesia species infecting sheep in Sudan, Ann. Parasitol. 62 (1) (2016) 47–54. [17] G. Uilenberg, Babesia?a historical overview, Vet. Parasitol. 138 (1-2) (2006) 3–10. [18] P. Parola, C.D. Paddock, C. Socolovschi, M.B. Labruna, O. Mediannikov, T. Kernif, et al., Update on tick-borne rickettsioses around the world: a geographic approach, Clin. Microbiol. Rev. 26 (4) (2013) 657–702. [19] Juckett G. Arthropod-Borne Diseases: The Camper’s Uninvited Guests, Microbiol. Spectr. 3 (4) (2015). [20] A.R. Beyer, J.A. Carlyon, Of goats and men: rethinking anaplasmoses as zoonotic infections, Lancet Infect. Dis. 15 (6) (2015) 619–620.
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Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria Atef Aouadi a,b , Hamza Leulmi c,d , Mehdi Boucheikhchoukh a , Ahmed Benakhla a , Didier Raoult c , Philippe Parola c,∗ a
Université Chadli Bendjdid, Département des Sciences Vétérinaires, El Tarf, 36000, Algeria Université Mohamed Cherif Messaadia, Institut des Sciences Agronomiques et Vétérinaires, Souk Ahras, 41000, Algeria c Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar), Inserm 1095, Marseille, France d Ecole Nationale Supérieure Vétérinaire d’Alger. El Aliya Alger, 16000, Algeria b
a r t i c l e
i n f o
Article history: Received 9 August 2016 Received in revised form 26 October 2016 Accepted 13 November 2016 Keywords: Theileria ovis Babesia ovis Small ruminants Ticks Algeria
a b s t r a c t Using qPCR, standard PCR and/or sequencing, we investigated the presence of tick-associated microorganisms in ticks and blood from sheep and goats from Souk Ahras, Algeria. Borrelia theileri, was detected in (7/120, 5.8%) blood from sheep and (13/120, 10.8%) goats. Anaplasma ovis was screened in (38/73, 52%) Rhipicephalus bursa and (5/22, 22.7%) R. turanicus and in (74/120, 61.7%), (65/120, 54.2%) blood of sheep and goats respectively. Coxiella burnetii tested positive in R. bursa (4/73, 5.5%) and (7/120, 5.8%) blood of sheep and (2/120, 1.7%) goats. Theileria ovis was detected in (50/147, 34%) R. bursa and (3/22, 13.6%) R. turanicus and in (64/120, 53.3%) blood of sheep and (25/120, 20.8%) goats. Babesia ovis was screened positive in (23/147, 15.6%) R. bursa and (7/48, 14.6%) R. turanicus. Our findings expand knowledge about the repertoire of tick-borne microorganisms present in ectoparasites and/or the blood of small ruminants in Algeria. © 2016 Published by Elsevier Ltd.
1. Introduction Small ruminants are an important source of meat and milk in Algeria and play a vital role in food security. Sheep predominate, at around 80% of total livestock. They are amongst the major economically important livestock in Algeria with about 27 million head in 2014 [1,2]. Goat populations also play an important role in the livelihood of resource-poor farmers and represent around 4 million head [2]. Sheep and goats are exposed to several health problems and their productivity is thought to be greatly reduced due to infectious diseases and parasitic diseases [2–5]. Vector-borne diseases represent a large proportion of these infectious diseases, which are caused by a multitude of pathogens transmitted by arthropod vectors [6]. Ticks were the first arthropods to be established as vectors of pathogens [7] that can transmit
∗ Corresponding author at: Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses Tropicales et Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, Faculté de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France. E-mail address: [email protected] (P. Parola). http://dx.doi.org/10.1016/j.cimid.2016.11.008 0147-9571/© 2016 Published by Elsevier Ltd.
them to humans [8]. They are considered to be the second biggest vectors of pathogens after mosquitoes [9] and the most important vectors of disease-causing pathogens in animals [10]. There are about 900 species of ticks described so far [11], most of which are responsible for the transmission of a huge variety of microorganisms, including viruses, bacteria and parasites [12]. Theileria spp. and Babesia spp., the causative agents of theileriosis and babesiosis, respectively, are among the most economically important hemoparasitic tick-borne diseases in ruminants worldwide [13]. In the past, most attention has been given to bovine-infecting piroplasmosis, and most tick-borne diseases in small ruminants have received less consideration [14]. However, because of the socio-economic impact in a number of countries, interest has recently increased in sheep and goat piroplasmosis [14]. The Theileria species infecting small ruminants are Theileria ovis, T. lestoquardi, T. luwenshuni, T. uilenbergi, T. recondita and T. separata [15,16]. Small ruminants are also affected by Babesia ovis, B. motasi, B. crassa, B. foliata, B. taylori, Babesia sp. (China) and Babesia sp. (Xinjiang) [17]. Likewise, a vast number of tick-related bacteria have been identified in the past 25 years [18], including borrelioses [19],
10 (5 b + 5 t) 10 (5 b + 5 t) 10 (5 b + 5 t) 00 10 (10 b) 00 10 (7 b + 3 t) 10 (10 b) 10 (8 b + 2 t) 10 (10 b) 10 (8 b + 2 t) 05 (5 b)
From goats From sheep R. turanicus R. bursa
35 (28 f + 7 m) 3 (1 f + 2 m) 54 (48 f + 6 m) 00 10 (8 f + 2 m) 2 (1 f + 1 m) 18 (3 f + 15 m) 00 19 (14 f + 5 m) 00 18 (14 f + 4 m) 10 (5 f + 5 m) 68 (33 f + 35 m) 4 (3 f + 1 m) 00 00 00 00 28 (3 f + 25 m) 2 (2 f) 00 00 12 (3 f + 9 m) 00 283 6/10 7/10 4/10 3/10 2/10 3/10 6/10 00/10 00/10 4/10 00/10 3/10 f; female, m; male, b; Rhipicephalus bursa, t; Rhipicephalus turanicus
R. turanicus R. bursa
75 (55 f + 20 m) 29 (11 f + 18 m) 95 (72f + 23 m) 16 (9 f + 7 m) 129 (110 f + 19 m) 30 (8 f + 22 m) 00 00 87 (80 f + 7 m) 00 00 00 82 (80 f + 2 m) 3 (3 f) 35 (20 f + 15 m) 00 10 (8 f + 2 m) 2 (2 m) 17 (5 f + 12 m) 00 10 (8 f + 2 m) 2 (2 m) 5 (5 f) 00 627 104 111 159 00 87 00 85 35 12 17 12 05 627 10/10 10/10 9/10 00/10 5/10 00/10 6/10 3/10 4/10 2/10 3/10 1/10
Ticks species and sex Total of tick collected from sheep Infested sheep /total of sampled sheep Sites of the study
Table 1 Ticks collection and blood sampling.
This cross-sectional study was carried out on ticks and blood from sheep and goats on 12 sites (Table 1) through the province of Souk Ahras in the Northeastern of Algeria, (36◦ 17 15 N 7◦ 57 15 E). Souk Ahras is surrounded by wooded mountains and is 1000 m above sea level. It is an extension of the Telli Atlas mountains. It has a semi-humid climate characterized by a hot summer and a cold, wet winter with rainfall averaging 850 mm per year, the bulk of which is seen between October and April. Small ruminants in all farms in the study were kept under a semi-extensive system, characterized by free grazing on pastures. Sheep and goats were kept under semi-enclosed systems at night and allowed to graze on pastures where the animals mixed with cattle, dogs and horses grazing in the same area. Twelve farms throughout the province of Souk Ahras were included in this study according to the cooperation degree of their owners. Ten sheep and 10 goats were randomly selected and sampled per farm. A total of 120 Barbarine sheep blood and 120 Arbi goat blood samples were collected individually in EDTA Vacutainer tubes from small ruminants. This cross-sectional survey was done in April 2014 and June 2015, periods when ruminants graze in pastures and are exposed to ticks and tick-borne diseases. With oral permission from all of the animals’ owners, blood samples were taken from apparently healthy sheep and goats and collected by cephalic venipuncture into tubes. The blood samples were collected from randomly selected male and female small ruminants including adults and yearlings. Once the blood was sampled, the entire body of each of the 240 animals was inspected for ticks. In order to reduce the time of restraint and handling of the animal we focused particularly on the ears, neck, udders and external genitalia (predilection sites of the ticks attachment). Ticks were removed manually and using blunted clockmakers’ forceps and immediately placed in 70% ethanol inside tubes labeled with the identification number and the date of collection. Only a portion of the collected ticks was used for the present study. All biological materials including blood and ticks were forwarded to Marseille, France, for morphological identification of ticks at the species level using morphological criteria within standard taxonomic keys [30], and stored at −20 ◦ C prior to molecular analyses (both blood and ticks).
Infested goats/ total of sampled goats
2.1. Study areas, tick collection and blood sampling
Ouilen Sedrata Khedara Mechrouha Merahna Hedadda Souk Ahras Medaourouch Oum Adayem Oued Kabarite Taraguelte Henancha Total
2. Materials and methods
38 54 12 18 19 28 72 00 00 30 00 12 283
Total of tick collected from goats
Ticks species and sex
Total tick qPCR tested
anaplasmosis [20], Q fever [21] and tick-borne rickettsial diseases [18]. In recent years, the prevalence of tick-borne bacterial diseases has dramatically increased and emerged throughout the world [22]. Spirochaete Borrelia theileri has been identified in the blood of various domestic mammals including ongulates, mainly cattle and small ruminants in tropical and subtropical regions of Africa, where the genus Boophilus (Rhipicephalus) was recognized as vector [23]. The clinical signs of infection in ruminants and other animals remain mild and variable, but usually include fever and anemia [24]. Anaplasma ovis is an erythrocytic anaplasma that parasitizes red blood cells [25] and may cause mild to severe disease in sheep and other ruminants [26]. A. ovis is transmitted primarily by Rhipicephalus bursa ticks [27]. In addition, Coxiella burnetii, the causative agent of Q fever, is recognized as a worldwide highly infectious zoonotic intracellular bacterium [28]. C. burnetii is able to infect various species of domestic mammals, including small ruminants and humans [29]. In this study, we aimed to update the occurrence and repertoire of tick-borne parasitic and bacterial diseases in Algeria by using molecular-based methods.
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10 (7 b + 3 t) 10 (10 b) 10 (8 b + 2 t) 10 (10 b) 10 (10 b) 10 (5 b + 5 t) 10 (6 b + 4 t) 00 00 10 (8 b + 2 t) 00 10 (10 b) 185
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2.2. DNA extraction Prior to DNA extraction, a convenient sample of ticks was selected according to a good representation of species and hosts, however all blood samples were processed to extract their DNA. All experiments and handling of blood and ticks were conducted in a laminar flow biosafety hood. A total of 200 L of blood was transferred to a specific tube prior to extraction using the QIAamp tissue kit (Qiagen, Hilden, Germany) and EZ1 robot as described by the manufacturer. Each ethanol-conserved tick was rinsed twice in sterile water for 10 min and dried on filter paper. Each sample was then longitudinally incised using an individual scalpel into two parts, one on which was crushed in sterile tubes (Eppendorf; Hamburg, Germany). The remaining portion of each tick was kept at −80 ◦ C for further control. Each tick sample was incubated overnight at 56 ◦ C in 180 L of buffer G2 and 20 L of proteinase K for pre-lysis followed by extraction using QIAamp tissue kit (Qiagen, Hilden, Germany) and EZ1 robot. For all samples (ticks and blood), the final elution volume was 100 L. 2.3. Molecular tests Once DNA had been extracted, it was used in real time PCR template assays to detect bacteria (Borrelia spp., Anaplasma spp., Coxiella burnetii, Bartonella spp. and Rickettsia spp.) and protozoan parasites (Theileria spp. and Babesia spp.). The qPCR reaction mixture consisted of 5 L of DNA and 15 L of mix from the Takyon PCR Kit (Qiagen, Hilden, Germany) as described [3]. Two negative controls were used in each qPCR plate and consisted of 5 L DNA extracted from uninfected ticks from our laboratory colony and 15 L takyon mix. Positive controls included DNA extracted from a positive samples for Theileria spp. and Babesia spp. and dilution of cultured strains of Borrelia crocidurae (for the detection of Borrelia spp.), Anaplasma phagocytophylum (for the detection of Anaplasma spp.), Coxiella burnetii (for the detection of C. burnetii), B. elizabethae (for the detection of Bartonella spp.) and R. montanensis (for the detection of Rickettsia spp.). Results were deemed positive if the cycle threshold value obtained by CFX96TM was lower than 35. All positive results were confirmed with a second qPCR system and/or sequence reaction. 2.3.1. Detection of Borrelia spp All DNA samples (ticks and blood) were individually tested for the presence of Borrelia spp. using the Bor16S system, which targets the rrs gene that encodes the 16S subunit of Borrelia genus-specific as described elsewhere [31]. Samples positive for Borrelia spp. by qPCR were further subjected to standard PCR to amplify a portion of the flaB (flagellin) and 16S rRNA genes of Borrelia spp. as described previously [31]. The success of the PCR amplification was verified by 1.5% of Agarose gel migration, with ethidium bromide at 130 V for 25 min. The bands were visualized under ultraviolet illumination using BenchTop pGEM® DNA Marker (Promega, Madison, Wisconsin, USA) to estimate the size of the products. The PCR products were then purified using the NucleoFast 96 PCR plate MacheryNagel EURL, France, as recommended by the manufacturer. The obtained amplicon was sequenced in the ABI 31000 automated sequencer (Applied Biosystems) by using the DNA sequencing BigDye Terminator Kit (Perkin- Elmer, Waltham, MA) as described by the manufacturer. The sequences obtained were assembled using ChromasPro 1.7 (Technelysium Pty Ltd., Tewantin, Australia) and compared with borrelias sequences in the GenBank database using BLAST: (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE TYPE=BlastSearch&LINK LOC=blasthome).
2.3.2. Detection of Anaplasma spp. All samples were primarily screened by qPCR to determine the presence of Anaplasmataceae in the DNA from tick and blood samples. Primers and a Taqman® probe targeting the 23S rRNA gene were used to amplify an amplicon of 280 bp as described elsewhere [32]. Positive results were confirmed by standard PCR and sequenced as described in the Detection of Borrelia spp. section using two systems of standard PCR targeting 16S Ehr and 23S [32]. 2.3.3. Detection of Coxiella burnetii As a first step, C. burnetii bacterial DNA was detected by qPCR system with C. burnetii–specific primers and a probe designed to amplify the IS1111 gene spacer [33]. Next another qPCR that amplified the IS30a spacer was used to confirm C. burnetii–positive results [3]. 2.3.4. Detection of Rickettsia spp. All the extracted DNA was assessed for the presence of rickettsial DNA by using a Rickettsia genus-specific qPCR for spotted-fever group Rickettsia by targeting the gltA gene (RKND03 system) [34]. 2.3.5. Detection of Bartonella spp. All DNA samples from ticks and blood were individually tested for the presence of the 16S–23S ribosomal RNA intergenic spacer gene by qPCR with a 21-base pair probe specific for the genus Bartonella as described [3]. 2.3.6. Detection of Theileria spp. and Babesia spp. Initially all samples were screened by qPCR targeting a fragment of the 28S to detect Theileria spp. using the following set of primers (Thei 28s F: 5 -NTACAGACTCAAAGGATCGATAGGC-3 and Thei 28s R: 5 - AAGGCGAACTCAATGAGGACA-3 ) and the probe (Thei 28s P: 6FAM- TCAAGCGAACTTTTCCCCTTTTGGTC-TAMRA). The presence of Babesia spp. DNA in ticks and blood samples was also determined by qPCR targeting 18S using the following primers (Bab 18s F: 5 -TTGGGGGCATTCGTANTNRAC-3 and Bab 18s R: 5 -TTCTTGATTAATGAAAACGTCTTG-3 ) and the probe (Bab 18s P: 6FAM-AAGACGAACTACTGCGAAAGCATTTGC-TAMRA). We designed both systems to detect Theileria spp. and Babesia spp. in our laboratory. Positive results were confirmed and sequenced by standard PCR targeting 18S rRNA. The 18S ribosomal RNA gene was amplified by PCR, with a pair of generic apicomplexan 18S rRNA-specific primers: CRYPTOF, the forward primer (5 AACCTGGTTGATCCTGCCAGT-3 ), and CRYPTOR, the reverse primer (5 GCTTGATCCTTCTGCAGGTTCACCTAC-3 ) as described elsewhere [35]. The conditions for the standard PCR included 95 ◦ C for 15 min, followed by 39 cycles of denaturation at 94 ◦ C for 30 s, annealing at 65 ◦ C for 30 s, and extension at 72 ◦ C for 1.5 min. The final extension was done at 72 ◦ C for 9 min, followed by a hold step at 15 ◦ C [36]. 3. Results 3.1. Tick identification and sample collection A total of 910 ticks were collected from sheep (627/910, 68.9%) and goats (283/910, 31.1%) from the province of Souk Ahras. Two tick species were identified among overall collected ticks: Rhipicephalus bursa (545/627, 86.9%) and Rhipicephalus turanicus (82/627, 13.1%), collected from 120 sheep and R. bursa (262/283, 92.6%) and R. turanicus (21/283, 7.4%), collected from 120 goats. From all the ticks collected, a convenient sample of 95 ticks sampled on sheep (73 R. bursa and 22 R. turanicus) and 90 ticks collected on goats (74 R. bursa and 16 R. turanicus) were PCR processed in this
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Table 2 Detection of ticks borne micro-organisms in blood and ticks from sheep, goats. Sites of the study Ouilen Sedrata Khedara Mechrouha Merahna Hedadda Souk Ahras Medaourouch Oum Adayem Oued Kabarite Taraguelte Henancha Total
Borrelia theileri
Anaplasma ovis
Coxiella burnetii
Theileria ovis
Babesia ovis
– 2 s, 5 g – 5g 3 s, 2 g – – – – – 1g 4s 7 s, 13 g
8 s, 8 g, 1 b 6 s, 5 g, 2 b, 2 t 5 s, 2 g, 3 b, 3 t 6 s, 9 g 6 s, 5 g, 7 b 3 s, 5 g 7 s, 1 g, 5 b 6 s, 8 g, 9 s, 2 g, 7 b 8 s, 9 g, 8 b 4 s, 6 g, 3 b 6 s, 5 g, 2 b 74 s, 65 g, 38 b, 5 t
– 2s 3 s, 4 b – 2s 2g – – – – – – –
8 s, 4 g, 3 b 2 s, 4 b 5 s, 6 g, 3 t, 2 Rb 3 s, 2 g, 2 Rb 5 s, 4 g, 7 b 5 s, 2 g 1 s, 6 b, 1 Rb 5 s, 4 g, 6 b 9 s, 6 s 7 s, 6 b, 2 Rb 6 s, 7 b 8 s, 3 g, 4 b 64 s, 43 b,
1 t, 5 Rb – 4 b, 2 t, 2 Rt – – – 5 Rb – – 7b – 2 b, 2 t –
b; Rhipicephalus bursa collected from sheep, Rb; Rhipicephalus bursa collected from goats, t; Rhipicephalus turanicus collected from sheep, Rt; Rhipicephalus turanicus collected from goats, s; blood of sheep, g; blood of goats, -; negative
study, with 240 blood samples from 120 sheep and 120 goats, to detect bacteria and protozoan parasites.
ples showed that the closest sequences available in GenBank were those for Theileria ovis: accession nos. FJ603460.1, AY508457.1, AY508456.1, AY260172.1 and EU622911.1.
3.2. Detection of bacteria 3.2.1. Detection of Borrelia spp. Of the 185 qPCR tested ticks, no DNA of Borrelia spp. was detected, however (7/120, 5.8%) sheep blood and (13/120, 10.8%) goat blood were positive using the 16S qPCR system from the Borrelia genus (Table 2). DNA sequence analyses of the PCR products targeting the 16S and flab on the Borrelia blood positive showed 100% similarity with Borrelia theileri (GenBank accession no. KF569941.1, 1206/1206 bp), regardless of the origin. 3.2.2. Detection of Anaplasma spp. Overall 23S qPCR tested ticks, (38/73, 52%) R. bursa and (5/22, 22.7%) R. turanicus collected on sheep were positive (Table 2). Anaplasma spp. was also screened positive in sheep blood (74/120, 61.7%) and goat blood (65/120, 54.2%). Sequencing analysis using the 16S Ehr system was performed on Anaplasma spp. positive ticks (10 samples) and blood (10 samples) and was not discriminating among Anaplasma ovis, A. marginale, and A. centrale, showing 100% similarity with these three Anaplasma species. The sequencing results using the 23S standard PCR system showed that all sequences of Anaplasma spp. detected in ticks and blood were similar to the sequence of Anaplasma ovis (Anaplasma ovis strain KMND Niayes 14 23S ribosomal RNA gene, partial sequence, GenBank accession no. KM021411.1). 3.2.3. Detection of Coxiella burnetii DNA from C. burnetii was detected in R. bursa (4/73, 5.5%) collected on sheep, blood of sheep (7/120, 5.8%) and blood of goats (2/120, 1.7%) (Table 2). We confirmed the result using a second qPCR system. 3.2.4. Detection of Rickettsia spp. and Bartonella spp. All the selected 185 ticks, 120 sheep and 120 goat blood samples tested negative for Rickettsia spp. and Bartonella spp. 3.3. Detection of haemoprotozoan-parasites 3.3.1. Detection of Theileria spp. Of the 185 tested ticks, 53 samples; (43/73, 58.9%) R. bursa and (3/22, 13.6%) R. turanicus collected on sheep, and (7/74, 9.4%) R. bursa sampled on goats, were positive for genus-specific Theileria by qPCR. Sixty-four sheep and 25 goat blood samples were also positive using the Theileria-qPCR system (Table 2). Subsequent sequencing of the 18S gene amplicons from all positive PCR sam-
3.3.2. Detection of Babesia spp. Overall in ticks and blood qPCR tested, Babesia spp. was screened positive in (13/73, 17.8%) R. bursa and (5/22, 22.7%) R. turanicus collected on sheep, and (10/74, 13.5%) R. bursa and (2/26, 7.7%) R. turanicus sampled on goats (Table 2). No blood was Babesia-qPCR positive. The amplification with sequencing of a fragment of the 18S gene of these Babesia positive samples showed 100% similarity with Babesia ovis accession nos. AY260178.1 and AY150058.1. 4. Discussion This investigation reports the first direct evidence of tick-borne haemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria (Anaplasma ovis, Borrelia theileri and Coxiella burnetii) in ticks (R. bursa and/or R. turanicus) and blood from small ruminants (sheep and goats) in Souk Ahras, Northern Algeria. In our study, the presence of DNA from pathogens in ticks and blood collected from small ruminants was screened using a validated method consisting of double qPCR assays and/or subsequent sequencing methods. The robustness of the data that we report is based on strict laboratory procedures and controls, including rigorous positive and negative controls to validate each test. Vector-borne agents are increasingly recognized as important causes of morbidity and mortality in humans and domestic animals worldwide [37]. In addition, in Algeria, the incidence of tick-borne zoonoses has increased over the last decade [3,31,38]. To the best of our knowledge, this is the first molecular study demonstrating the presence of piroplasms, T. ovis and B. ovis, in ticks and blood from small ruminants in Algeria. Our work on Theileria spp. was carried out on 120 sheep blood samples, 120 goat blood samples and 185 ticks, in which we found 64 (53.3%) sheep, 25 (20.8%) goats and 53 (28.5%) Rhipicephalus ticks that were T. ovis DNA positive. Initially, T. ovis, was considered to be less pathogenic than T. lestoquardi, the agent of malignant ovine theileriosis, which is the major pathogen present in the arid regions of Africa [39]. Until recently, T. ovis was identified mainly by its non-pathogenicity for sheep and goats [14], which could explain the absence of reported clinical theileriosis cases in the studied regions. T. ovis was assessed as positive via reverse-line blotting in R. turanicus ticks and blood collected from sheep and goats from Tunisia [40]. T. ovis from the Mediterranean basin has R. bursa as vector, where it was detected in sheep and goats from Southern Catalonia, Spain [41].
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Babesia ovis was detected in our study, in R. bursa and R. turanicus ticks sampled on sheep and goats. B. ovis, unlike T. ovis, is the causative agent of ovine babesiosis with B. motasi and B. crassa [42]. B. ovis is highly pathogenic to sheep and goats and its pathogenicity is well known [40]. Clinical symptoms of the disease include fever, anemia, loss of appetite, hemoglobinuria, icterus, and death [42]. The R. bursa tick is the principal vector of B. ovis in Mediterranean areas [43] where ovine babesiosis is prevalent, mainly in North Africa [40]. In addition to hemoparasites detected in this investigation, tickborne diseases were also screened positive in our study. We identified the DNA of the spirochaete Borrelia theileri in blood of sheep and goats. B. theileri is the causative agent of bovine borreliosis in cattle [24] and considered to be one of the relapsing fever Borreliae that have been related as being hosted by hard ticks [37]. Rhipicephalus spp. is the main recognized vector in several countries of the world, mainly in tropical and subtropical regions of Africa [23;24]. B. theileri was detected from Boophilus microplus and Rhipicephalus geigyi in Mali [44] and showed 100% similarity with the B. theileri that we found. Generally the clinical signs of infection caused by B. theileri are variable and mild, usually associated with fever and anemia [24]. In this study, Anaplasma ovis was also molecularly identified in the blood of sheep and goats and in R. turanicus and R. bursa ticks sampled on sheep. A. ovis is the causative agent of anaplasmosis in small ruminants, where it invades and propagates within their erythrocytes, as are A. marginale and A. centrale [25]. Clinical signs may be mild to severe [26]. A. ovis is known to be transmitted by R. bursa ticks [27]. In Algeria, there is a lack of information about the Anaplasmataceae infection in animals and mainly in ruminants, where only A. phagocytophilum, A. platys and Anaplasma sp. were found in cattle [32]. Finally, we detected the agent of Q fever, C. burnetii, in R. bursa ticks and blood from sheep and goats. C. burnetii is a highly infectious zoonotic intracellular bacterium [28] that can affect different species of wild and domestic mammals [29]. Q fever has been described worldwide in outbreaks involving sheep, goats, cats, dogs and wild animals, while reservoirs are extensive but only partially known and include mammals, birds, and arthropods, mainly ticks [45]. In Algeria a few human cases of Q fever have been documented, with only two human cases reported in Oran [46]. DNA from C. burnetii was recently reported in ticks from bats in Souk Ahras, Algeria [3] and in dog and cat spleens [47]. Overall our study, we detected Borrelia theileri only in blood of sheep and goats. In opposite, Babesia ovis was screened positive only in ticks of sheep and goats. In addition, Anaplasma ovis, Coxiella burnetii and Theileria ovis DNA were present in ticks and in blood of sheep and goats. In our case, mainly, the positive animals have a positive ticks. In case, if ticks were removed from their hosts, that it could have been bacteremic or parasitemic, the DNA of the detected micro-organism can be present in ticks and in animal blood simultaneously and ticks can be vectors for some pathogens or simple carriers in other cases [3]. However, if the animals is not bacteremic, or parasitemic or the ticks have been infected through the blood of other infected animals and newly attached to a new animal host, in this case ticks can be positive and the animal host not. To date, information about vector-borne diseases agents circulating in Algeria remains limited mainly to hemoparasitic diseases. This paper provides interesting new information on zoonotic pathogens emerging from small ruminants in Northern Algeria. Our findings will help human and veterinary clinicians to enlarge the panel of pathogens to consider in differential diagnosis. Future epidemiological studies using larger numbers of domestic animals and ectoparasites from more localities is necessary in order to determine the actual prevalence and distribution of these diseases in
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