Molecular screening and genetic diversity of tick-borne pathogens associated with dogs and livestock ticks in Egypt

Abstract

Background: The Middle East and North Africa (MENA) offer optimal climatic conditions for tick reproduction and dispersal. Research on tick-borne pathogens in this region is scarce. Despite recent advances in the characterization and taxonomic explanation of various tick-borne illnesses affecting animals in Egypt, no comprehensive examination of TBP (tick-borne pathogen) statuses has been performed. Therefore, the present study aims to detect the prevalence of pathogens harbored by ticks in Egypt.

Methodology/principal findings: A four-year PCR-based study was conducted to detect a wide range of tick-borne pathogens (TBPs) harbored by three economically important tick species in Egypt. Approximately 86.7% (902/1,040) of the investigated Hyalomma dromedarii ticks from camels were found positive with Candidatus Anaplasma camelii (18.8%), Ehrlichia ruminantium (16.5%), Rickettsia africae (12.6%), Theileria annulata (11.9%), Mycoplasma arginini (9.9%), Borrelia burgdorferi (7.7%), Spiroplasma-like endosymbiont (4.0%), Hepatozoon canis (2.4%), Coxiella burnetii (1.6%) and Leishmania infantum (1.3%). Double co-infections were recorded in 3.0% (27/902) of Hy. dromedarii ticks, triple co-infections (simultaneous infection of the tick by three pathogen species) were found in 9.6% (87/902) of Hy. dromedarii ticks, whereas multiple co-infections (simultaneous infection of the tick by ≥ four pathogen species) comprised 12% (108/902). Out of 1,435 investigated Rhipicephalus rutilus ticks collected from dogs and sheep, 816 (56.9%) ticks harbored Babesia canis vogeli (17.1%), Rickettsia conorii (16.2%), Ehrlichia canis (15.4%), H. canis (13.6%), Bo. burgdorferi (9.7%), L. infantum (8.4%), C. burnetii (7.3%) and Trypanosoma evansi (6.6%) in dogs, and 242 (16.9%) ticks harbored Theileria lestoquardi (21.6%), Theileria ovis (20.0%) and Eh. ruminantium (0.3%) in sheep. Double, triple, and multiple co-infections represented 11% (90/816), 7.6% (62/816), and 10.3% (84/816), respectively in Rh. rutilus from dogs, whereas double and triple co-infections represented 30.2% (73/242) and 2.1% (5/242), respectively in Rh. rutilus from sheep. Approximately 92.5% (1,355/1,465) of Rhipicephalus annulatus ticks of cattle carried a burden of Anaplasma marginale (21.3%), Babesia bigemina (18.2%), Babesia bovis (14.0%), Borrelia theleri (12.8%), R. africae (12.4%), Th. annulata (8.7%), Bo. burgdorferi (2.7%), and Eh. ruminantium (2.5%). Double, triple, and multiple co-infections represented 1.8% (25/1,355), 11.5% (156/1,355), and 12.9% (175/1,355), respectively. The detected pathogens' sequences had 98.76-100% similarity to the available database with genetic divergence ranged between 0.0001 to 0.0009% to closest sequences from other African, Asian, and European countries. Phylogenetic analysis revealed close similarities between the detected pathogens and other isolates mostly from African and Asian countries.

Conclusions/significance: Continuous PCR-detection of pathogens transmitted by ticks is necessary to overcome the consequences of these infection to the hosts. More restrictions should be applied from the Egyptian authorities on animal importations to limit the emergence and re-emergence of tick-borne pathogens in the country. This is the first in-depth investigation of TBPs in Egypt.

Fig 1. Egypt’s map showing the tick collection spots and their distribution in the study area.

Green color refers to Egypt, black to Alexandria governorate, violet to Beheira governorate, and red to Marsa Matrouh governorate. Brown map marks refers to collection sites of Hyalomma dromedarii, light blue map marks to Rhipicephalus annulatus, and yellow map marks to Rhipicephalus rutilus. Open Street Map, which is licensed under an Open Database License ODbL 1.0, was used. The base layer was extracted here: https://www.openstreetmap.org/export. The terms and conditions of the copyright are provided here: https://www.openstreetmap.org/copyright. https://doi.org/10.1371/journal.pntd.0012185.

Fig 2. Maximum-likelihood tree based on sequences of the 16S rRNA gene of Anaplasma species detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Tamura-Nei model using Ehrlichia chaffeensis as an outgroup.

Fig 3. Maximum-likelihood tree based on sequences of the 18S rRNA gene of Babesia species detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the General Time Reversible (GTR) model using Plasmodium vivax as an outgroup.

Fig 4. Maximum-likelihood tree based on sequences of the 16S rRNA gene of Rickettsia species detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Kimura 2-parameter model using Listeria monocytogenes as an outgroup.

Fig 5. Maximum-likelihood tree based on sequences of the 18S rRNA gene of Theileria detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Hasegawa-Kishino-Yano model using Cytauxzoon felis as an outgroup.

Fig 6. Maximum-likelihood tree based on sequences of the 16S rRNA gene of Mycoplasma/Spiroplasma detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Tamura-Nei model using Kareius bicoloratus as an outgroup.

Fig 7. Maximum-likelihood tree based on sequences of the 16S rRNA gene of Ehrlichia canis detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Kimura 2-parameter model using Neisseria weaveri as an outgroup.

Fig 8. Maximum-likelihood tree based on sequences of the ribonuclease III (pCS20) gene of Ehrlichia ruminantium detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Hasegawa-Kishino-Yano model using Pseudomonas sp. as an outgroup.

Fig 9. Maximum-likelihood tree based on sequences of the 5S-23S rRNA intergenic spacer of Borrelia burgdorferi detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Tamura 3-parameter model using Borrelia americana as an outgroup.

Fig 10. Maximum-likelihood tree based on sequences of the FlaB gene of Borrelia theileri detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Tamura 3-parameter model using Leptospira interrogans as an outgroup.

Fig 11. Maximum-likelihood tree based on sequences of the 16S rRNA gene of Coxiella burnetii detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Hasegawa-Kishino-Yano model using Yersinia pestis as an outgroup.

Fig 12. Maximum-likelihood tree based on sequences of the 18S rRNA gene of Hepatozoon canis detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the General Time Reversible (GTR) model using Babesia equi as an outgroup.

Fig 13. Maximum-likelihood tree based on sequences of the ITS1 gene of Leishmania infantum detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Tamura 3-parameter model using Trypanosoma cruzi as an outgroup.

Fig 14. Maximum-likelihood tree based on sequences of the ITS1 gene of Trypanosoma evansi detected in the present study of Egyptian ticks (bold).

Numbers represent bootstrap support generated from 1,000 replications. The tree was constructed with the Kimura 2-parameter model using Leptomonas seymouri as an outgroup.