Evidence of the presence of Borrelia burgdorferi in dogs and associated ticks in Egypt

Background Borrelia burgdorferi is the spirochete that causes Lyme Borreliosis (LB), which is a zoonotic tick-borne disease of humans and domestic animals. Hard ticks are obligate haematophagous ectoparasites that serve as vectors of Borrelia burgdorferi. Studies on the presence of Lyme borreliosis in Egyptian animals and associated ticks are scarce. Methods This study was conducted to detect B. burgdorferi in different tick vectors and animal hosts. Three hundred animals (dogs=100, cattle=100, and camels=100) were inspected for tick infestation. Blood samples from 160 tick-infested animals and their associated ticks (n=1025) were collected and examined for the infection with B. burgdorferi by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. The identified tick species were characterized molecularly by PCR and sequencing of the ITS2 region. Results The overall tick infestation rate among examined animals was 78.33% (235/300). The rate of infestation was significantly higher in camels (90%), followed by cattle (76%) and dogs (69%); (P = 0.001). Rhipicephalus sanguineus, Rhipicephalus (Boophilus) annulatus, and both Hyalomma dromedarii and Amblyomma variegatum, were morphologically identified from infested dogs, cattle, and camels; respectively. Molecular characterization of ticks using the ITS2 region confirmed the morphological identification, as well as displayed high similarities of R. sanguineus, H. dromedarii, and A. Variegatu with ticks identified in Egypt and various continents worldwide. Just one dog (1.67%) and its associated tick pool of R. sanguineus were positive for B. burgdorferi infection. The 16S rRNA gene sequence for B. burgdorferi in dog and R. sanguineus tick pool showed a 100% homology. Conclusion Analyzed data revealed a relatively low rate of B. burgdorferi infection, but a significantly high prevalence of tick infestation among domesticated animals in Egypt, which possesses a potential animal and public health risk. Additionally, molecular characterization of ticks using the ITS2 region was a reliable tool to discriminate species of ticks and confirmed the morphological identification.


Background
Lyme disease (LD) or the tick-borne relapsing fever (TBRF) is an emerging tick-borne multi-systemic zoonotic bacterial disease that has a worldwide distribution, caused by spirochetes of the Borrelia burgdorferi group and transmitted by ticks of the Ixodes ricinus complex [1]. The bacterium is horizontally transmitted between ticks and within reservoir hosts of wild animals, mostly small mammals, and humans which are considered an incidental host [2]. Clinical signs differ according to the species of B. burgdorferi complex prevailing in the area, but most human patients show erythema migrans accompanied by flu-like symptoms [2,3]. Companion animals, particularly dogs, act as sentinels for Lyme disease [4]. Only 5-10% of infected dogs show clinical signs. Therefore, there is a significant underestimation of the Lyme disease prevalence in dogs, which represents a risk for disease spreading [5]. A previous survey on domestic animals detected borrelial DNA only in the adult ticks, mostly infesting sheep and cattle [6].
Ticks (subclass: Acari; order: Parasitiformes; suborder: Ixodida) are obligate haematophagous ectoparasites of wild and domestic animals and humans. Ticks are considered the most important vectors of diseasecausing pathogens within the phylum Arthropoda, being comparable only to mosquitoes (family Culicidae) [7,8]. As the incidence of tick-borne diseases propagates and expands geographically, it is becoming increasingly significant to distinguish tick species, to enhance tick and tick-borne disease control [9]. The brown tick Rhipicephalus sanguineus of dogs [10], the blue tick Rhipicephalus Boophilus of cattle [11], Hyalomma dromedarii of camels [12], and the tropical bont tick (TBT) Amblyomma variegatum of ruminants [13,14], are of significant veterinary and medical importance due to their vector competence for several pathogens [15].
Despite the existence of various hard tick species in Egypt, limited data on the borrelial infection of these ticks is available. Here, we studied the infection of the hard ticks in Egypt with B. burgdorferi. Additionally, we investigated the prevalence and phylogeny of Borrelia in the tick-infested dogs, cattle, and camels during the year 2017.
Phylogenetic analysis, based on the ITS2 region sequences of ticks, revealed relatedness of Egyptian ticks R. sanguineus, H. dromedarii, and A. Variegatu with other tick isolated from different continent all over the world, as shown in (Fig. 2).

Detection and phylogenetic analysis of B. burgdorferi
Borrelia burgdorferi was detected in a Rhipicephalus sanguineus tick collected from a dog that was also infected (Tables 1 and 2). Based on the 16S rRNA gene of B. burgdorferi collected from dog blood and ticks, a hundred percent of identity were reported between B. burgdorferi isolated from the blood of dogs (MH685928) and brown dog ticks (MH685927) in Egypt, as well as B. burgdorferi isolated from Ixodes pacificus in the USA (KY563172) (Fig. 3).

Discussion
Studies on Borrelia burgdorferi (Lyme disease) in Egypt are limited, especially in animals and tick vectors. Besides, the recent large-scale movements of humans and animals, as well as the increasing geographical distribution of several tick species, contributed to the growing global threat of tick-borne disease (TBD) [8,15]. Therefore, in the current study we moleculary identified the hard tick population infesting the examined animals, as well as, investigated the prevalence and phylogeny of B. burgdorferi in dogs, cattle, camels, and associated ticks.
Although the morphological identities of identified ticks were similar to the taxonomic key of Ixodidae ticks set by [20], further molecular identification of ticks was carried out through the examination of the ITS2 region. The choice of the ITS2 region was based on the previous studies [21,22],which indicated that this DNA marker is reliable in discriminating species of ticks. The sequence of the ITS2 region of our isolates showed varying degrees of similarities with local and international hard tick Those intra-species and inter-species similarities could be due to the agreements in the area of sample collection from northern Egypt as well as the same infested cattle population. Genetic identity of the Egyptian Hyalomma dromederii (MH685931) collected in this study from camels (Camelus dromedaries), proved a high sequence homology of 100% similarity with previous H. dromederii isolates from Matrouh in Egypt, with  [26] and 99.9% with one collected from wild herbivores in Kenya (KM819713). The detection of borrelial DNA in infested animals and associated ticks revealed that one dog (1.67%) and its associated pool of adult Rhipicephalus sanguineus ticks (1.67%) were infected with B. burgdorferi. The low tick infection rate with B. burgdorferi reported in our study disagrees with that recorded in Fayoum and Beni-Suif in Egypt, by [27] who reported 7/12 (58.33%) infected pools of R. sanguineus. This difference could be assigned to different geographical locations; as Cairo and Giza are more urbanized than Fayoum and Beni-Suif which are mostly rural governorates. The prevalence of B. burgdorferi in the examined dogs in Italy was 1.47%. In the USA, infection rates with B. burgdorferi recorded 1.2, 4.0, and 6.7% in dogs from three different regions [28,29]. The most recent investigations about the prevalence of B. burgdorferi epidemic in the European canine population exposed different rates, as the highest incidence observed in Poland 40.2% [30,31] and the lowest was in Portugal 0.2-0.5% [32] In a previous study [6] in Iran, researchers could detect borrelial DNA only in the adult ticks. They attributed the lack of Borrelia infection in nymphs to limited nymph samples as they were unable to catch the immature specimen.
In our study, the identity of B. burgdorferi that was isolated from the dog's blood (MH685928) and the associated brown tick pool of R. sanguineus (MH685927) in Egypt, as well as B. burgdorferi isolated from Ixodes pacificus in the USA (KY563172), were 100% in similarity. From the veterinary and public health points of view, scientists should be aware that ticks are active in broad climatic conditions, even greater than those expected. The current level of knowledge about LB risk and the risk related to tick bites is quite limited and underestimated.

Conclusion
In Egypt, the domestic animal population is markedly infested with the hard ticks, which threatens the animal and public health with potential tick-borne pathogens. Our data revealed that the camel tick H. dromedarii is the most prevalent tick in Egypt. The molecular taxonomy of tick species is going to be a standard approach for confirming the morphological identification of ticks. Molecular detection of borrelial DNA showed a relatively low rate of B. burgdorferi infection in dogs and associated ticks. However, dogs act as a potential sentinel carrier of Lyme disease. Since B. burgdorferi is of zoonotic importance, routine monitoring of domestic animals, adequate control measures, and increased awareness of the possible Lyme Borreliosis infections are required for consideration of this pathogen in the differential diagnosis of infectious diseases.

Ticks collection
A three hundred Dogs (stray and pet dogs), cattle (native breed), and camels (Camelus dromedaries), 100 animals each, from Cairo, Giza, Al-Buhayrah, and Matrouh governorates, were inspected for tick infestation, for 1 year (2017). Adult hard tick samples were collected and isolated from those naturally infested animals, as indicated in the (Table 3) and (Fig. 4). Ticks were pulled off manually from the animals by hands and with blunt point forceps, then placed in a sterile loosely capped plastic vials and transported to the laboratory in a dry icebox. In the laboratory, the ticks were identified and stored at − 20°C.

Blood sampling
Blood samples were collected from hard tick-infested animals, including dogs (n = 60), cattle (n = 50), and camels (n = 50) from some Egyptian governorates, which are shown in (Table 3) and (Fig. 4). The blood samples were collected in tubes coated with EDTA from the cephalic vein in dogs, and the Jugular vein in cattle and camels. All blood samples were transported to the laboratory in an icebox and stored at − 20°C until use.

Tick identification Morphological identification of ticks
Tick samples were kept at room temperature and then washed twice with sterile normal saline to remove excess particulate contamination from animal skin, rinsed once with 70% ethanol. They were mounted on slides and examined using a stereoscope microscope (BOECO, Germany). Adult ticks were identified into genera, species, and subspecies by using appropriate identification keys of morphological shapes [33]. About 20 ticks were taken from each dog and about 30-35 ticks were taken from each cattle or camel as a target number of ticks from each animal, we did not collect all the ticks infesting on each animal, which is considered a limitation of the study. The identified ticks were transferred to sterile vials and stored at − 20°C until processing.

Molecular identification of ticks
Ticks' DNA extraction DNA was extracted from each morphologically identified tick sample, after being crushed in a mortar with liquid nitrogen into small pieces, by using a DNA extraction kit (DNeasy Blood & Tissue Kit, QIAGEN; Germany) according to the manufacture's protocol.
PCR conditions was done according to Muruthi et al., 2016 [35]. Amplification reactions were visualized on a 1% agarose gel containing 0.5 μg/ml ethidium bromide (Sigma), while a 1 kb DNA ladder (Invitrogen™) was used as a marker. The gel was visualized under UV light with a transilluminator.

Sequencing of ticks' PCR products and phylogenetic analysis
PCR products of the identified ticks were purified from the reactions using the Qiaquick purification kit for tissues (Qiagen, Germany) according to the manufacturer's instructions. Sequencing was conducted using Big Dye Terminator V3.1 sequencing kit (Applied Biosystems) with forward primer ITS2 ribosomal DNA. Phylogenetic analysis was based on the ITS2 region sequences of ticks. The trees were constructed and analyzed by neighbor-joining.
Molecular detection of Borrelia burgdorferi 16S rRNA gene in blood and tick samples DNA extraction from blood and ticks DNA was extracted from blood samples using QIAamp DNA Blood Mini Kit (Qiagen, Germany), and from tick samples using QIAamp DNA Mini Kit for tissue (Qiagen, Germany) according to the manufacturer's instructions. The DNA extract was stored at − 20°C until being used in the PCR assay.
Sequencing of B. burgdorferi PCR product and phylogenetic analysis PCR products of positive B. burgdorferi samples (blood and ticks) were purified from the reactions using the Qiaquick purification kit (Qiagen, Germany) according to the manufacturer's instructions. Sequencing was conducted using Big Dye Terminator V3.1 sequencing kit (Applied Biosystems) with forward primer 16S rRNA. The obtained nucleotide sequence was compared with those available in public domains using NCBI, BLAST server. Sequences were downloaded and imported into BioEdit version 7.0.1.4 for multiple alignments using the Clustal W program of the BioEdit. Phylogenetic analysis was performed with MEGA version 7 using the neighbor-joining method. The bootstrap consensus tree was inferred from 950 replicates (Fig. 3). The phylogenetic tree was constructed based on the 16S rRNA gene sequences recovered from Borrelia burgdorferi strains isolated from different sources, including Dog (MH685928) and Tick (MH685927) and aligned with the other related 16S rRNA gene sequences obtained from GenBank NCBI-BLAST.

Statistics and spatial data
Analysis of data was performed with PASW Statistics, Version 18.0 software (SPSS Inc., Chicago, IL, USA). Data collected were analyzed using both the descriptive statistic (frequencies) and the Chi-square (χ 2 ) test for independence to examine the relation between animal species and tick infestation rates. A P-value < 0.05 was considered statistically significant. The map was created with ArcMap version 10.1 software.
The study was performed according to the guidelines of the ethical committee (Institutional Animal Care and Use Committee), Faculty of veterinary medicine, Cairo University, Vet CU. IACUC (VetCU1022019070).