Identification of hard tick species infesting camels at slaughterhouse and its potential role in transmitting Coxiella burnetii in Egypt

Background C. burnetii is an important pathogen because of its wide host range, low infectious dose, stability in the environment, and capability for aerosol dispersion. The aim of this work was to investigate the potential role of hard ticks found on camels in transmitting Coxiella burnetii. A total of 370 adult hard ticks were collected from 181 imported camels brought for slaughter in Cairo, Egypt. Ticks were identified using a stereomicroscope then screened for the presence of C. burnetii by PCR. Results Most camels were infested with Hyalomma dromedarii (n= 171, 94.5%) whereas other species were less prevalent, including Amblyomma hebraeum (10.5%), Rhipicephalus pulchellus (6.1%), H. anatolicum anatolicum (5%), A. variegatum (4.4%) and A. gemma (2.2%). It is important to note that A. variegatum , A. gemma and Rhipicephalus pulchellus are rarely identified in Egypt, despite the higher prevalence in countries where camels came from. Four out of the six identified ixodid tick species yielded positive results for C. burnetii with an overall prevalence of 5.4%, while prevalence of the other tick species was 6.6%, 5.6%, 5.3% and 3.6% for H. dromedarii , A. variegatum , H. anatolicum anatolicum and R. pulchellus respectively. Conclusion The current study identified a wide array of hard tick species found on camels and highlights the potential role of such ticks in transmitting C. burnetii .


Background
To date, ticks are the second biggest blood feeding arthropod of man and animals after mosquitoes [1]. They constitute a global problem by acting as vectors and reservoirs for several infectious pathogens of medical and veterinary importance [2]. For instance, C.
burnetii is transmitted to humans and animals through their bites. In man, C. burnetii is the causative agent of Q fever, an emerging and/or re-emerging zoonotic disease that has been reported all over the world [3]. The disease is usually manifested by fever, flu-like symptoms and pneumonia. Endocarditis may be observed in chronic cases.
In farm animals, tick infestation increases the susceptibility to infectious diseases and reduces milk and meat production [4]. Although C. burnetii is usually a sub-clinical disease, abortion may be the most important clinical outcome in ruminants [5].
Of note, the Ixodidae (hard ticks) is the largest tick family, which infests various animal hosts, domestic or wild and leads to the transmission of a wide array of pathogenic agents to mammalian hosts including man [6,7]. Globally,Somalia, Sudan, and to lesser extent Ethiopia are the main countries that contain the largest camel populations [8]. These countries export camels that usually live in arid areas [9]to Middle Eastern countries, with the transboundry transfer of their ticks and tick-borne pathogens.
There are more than 40 species of ticks that harbor C. burnetii. The bacterium is transmitted transstadially and transovarially among infected ticks [10,11].Ticks also shed huge numbers of C. burnetii in their feces (10 10 organisms per gram) to contaminate the environment. Thus, ticks play a vital role in maintaining C. burnetii in nature [12]. In addition, C. burnetii has numerous animal reservoirs comprising different mammals, birds, and arthropods [13].
In Egypt, there is a scarcity of knowledge in the taxonomic identification of tick species found on camels. They are brought to the abattoir for slaughter, with the great majority being imported rather than native. Because the potential role of such ticks in transmitting C. burnetii is fairly obscure, this study was carried out to give some insight into the diversity of tick species infesting camels in Egypt.

Tick collection and identification
A total of 370 adults ticks were gathered from 181 apparently healthy imported dromedary camels (Camelus dromedarius) that were admitted to Bassatin abattoir, Cairo, Egypt for slaughtering. Ticks were collected from the skin at the different predilection sites; head, brisket, belly, groin and tail. Each tick was carefully removed by sterile medium-sized forceps with blunt points, then inserted in sterile screw capped tubes. The collected ticks were transported in an icebox to the laboratory for identification. Upon arrival, ticks were examined under a stereobinocular microscope (BOECO, Germany) for morphological identification and classification into species level according to standard taxonomic guidelines for adult ticks as described by [14] (Fig.1). After completion of tick identification, ticks were stored at-20 °C till processing.
Molecular detection of C. burnetii among the examined ticks DNA extraction DNA from each adult tick was extracted using DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer's instructions. The extracted DNAs were stored at -20°C till use.
Extracted DNAs from ticks were tested for the presence of C. burnetii using primers trans-1 (5'-TAT GTA TCC ACC GTA GCC AGT C-3') and trans-2 (5'-CCC AAC AAC ACC TCC TTA TTC-3') to amplify a 687 bp fragment of the repetitive insertion element IS1111 of the transposase gene [15].
The PCR reaction volume was 25 µl and each reaction mixture contained 12.5 µl of 2X PCR HotStarTaq® master mix (Qiagen, Germany), 0.1μM of each primer, 3 µl of DNA template and completed up to 25 µl with nuclease free water. PCR assay was performed in T100™ Thermal Cycler (Bio-Rad, USA) under the following conditions: enzyme activation at 95 °C for 15 min followed by 5 cycles of denaturation at 94°C for 30 Sec, annealing at 66 to 61°C (the temperature was decreased by 1°C between consecutive steps) for 1 min and an extension at 72°C for 1 min. These cycles were followed by 40 cycles at 94°C for 30 Sec, 61°C for 30 Sec, 72°C for 1 min and then a final extension step of 10 min at 72°C.
C. burnetii DNA (Genekam, Germany) was used as a positive control, while nuclease free water was used as a negative control (Fig. 2).
DNA sequencing and phylogenetic analysis PCR products of three positive C. burnetii sampleswere purified using a QIAquick purification kit (Qiagen, Germany) for sequencing using Big Dye Terminator V3.1 kit (Applied Biosystems) in ABI 3500 Genetic Analyzer (Applied Biosystems, USA). Afterwards, the obtained sequences were compared with those available in the GenBank using a BLAST server on the NCBI website. Sequences were aligned against each other and compared to 2 sequences recorded in Egypt as well as some selected sequences isolated from human cases worldwide. The analysis was carried out using the Clustal W, BioEdit software (ver. 7.0.9). A neighbor-joining phylogenetic tree was constructed using Mega6.06 software and bootstrap analysis was obtained with 500 replicates (Fig. 3).

Nucleotide Sequence Accession Numbers
Three partial sequences of C. burnetii IS1111 gene were submitted to GenBank, which assigned them the following accession numbers:

Results
Six species of hard ticks were identified after examination of 181 camels enrolled in this study. The majority were found to be infested with H. dromedarii (94.5%), while other tick species were present in the following prevalence rates: Amblyomma hebraeum 10

Discussion
Hard ticks are common external parasites infesting camels, which may affect their health and productivity [16]. The results of the current study show that most of them were infested by H. dromedarii94.5%), in agreement with previous reports from Egypt [17,18] and Sudan [19]. It is known that H. dromedarii is a very characteristic tick closely associated with dromedary camels distributed in Africa [14]. Moreover, the current study identified a diversity of hard tick species on the examined camels, like A. gemma (2.2%), A. variegatum (4.4%) and R. pulchellus (6.1%). Such hard ticks are not geographically distributed in Egypt, but usually prevalent in Ethiopia, Somalia and Sudan, where these camels usually come from [20,16,21] Seriously, these exotic ticks may have great veterinary and public health implications as they are vectors for several emerging pathogens, including Crimean Congo hemorrhagic fever virus, Rickettsia Africae and C. burnetii [22,23,24]. Despite many studies conducted to investigate the prevalence of C. burnetii among ticks infesting different animals, a few investigated C. burnetii in ticks attached to camels. In the present study, C. burnetii was detected in an overall prevalence of 5.4%. This finding is almost comparable to that of [24] in Ethiopia (6.4%), [25] in Southeastern Iran (7.4%) and [26] in Malaysia (5.8%). C.
The results of this study show that the prevalence of C. burnetii among the examined H. dromedarii is 6.6%, which is considerably higher than that reported by [29] in Egypt (0.7%). H. dromedarii is known to harbor C. burnetii, which is shed in its feces and saliva [30]. Furthermore, C. burnetii was detected in H. anatolicum anatolicum at a prevalence rate of 5.3%, although [31] recorded a negative result after examination of 20 H. anatolicum ticks in northeastern Algeria. Also, we found that 5.6% of examined A. variegatum ticks were positive for C. burnetii; a result which is lower than that reported in Nigeria (19.6%) [32] and Senegal (37.6% [13], but higher than that recorded in Côte d'Ivoire (0.6%) [33], and Ethiopia (3.2%) [24]. Additionally, the examined R. pulchellus ticks yielded C. burnetii in a prevalence of 3.6%. These findings coincide with a those of [34], who detected C. burnetii in 3.03% of the investigated R. pulchellus ticks in Kenya, whereas [13] found the pathogen in 25% of examined ticks in Ethiopia.
None of the examined A. gemma and A. hebraeum showed positive results for C. burnetii, which is consent with the results of [35] in South Africa, but contradicts the findings of [13] in Ethiopia (28.6% prevalence).
There seems that A. variegatum and R. pulchellus that are common in Ethiopia, Somalia and Sudan are carried on camels coming to Egypt to appear at low rates, which raises a query about the impact of transboundary transmission and public health implications.
Moreover, the phylogenetic analysis of three randomly selected C. burnetii strains obtained in this study (MF197399-MF197401) revealed that 2 sequences were grouped in the same clade with C. burnetii strains isolated from febrile patients in Brazil, whereas the third sequence was placed in the same clade with that obtained from blood of patients in India and China (Fig.3). Such analysis highlights the public health significance of these strains. However, none of the obtained sequences were grouped in the same clade with the sequences reported in Egypt by [29,5]; a matter which suggests the genetic diversity or exotic nature of C. burnetii strains identified in this study.    Figure 1 The diversity of hard tick species attached to the examined camels (A1&A2) H. Phylogenetic analysis using neighbor-joining approach based on the partial sequence of the repetitive insertion element IS1111 of the transposase gene of C.

Conclusion
burnetii. The bootstrap consensus tree was constructed by Mega6.06 software to demonstrate the evolutionary history of the enrolled sequences.