Application of advanced molecular techniques to detect vector borne pathogens from stray dogs and cats in two different climatic zones of Saudi Arabia

Background : Vector-borne diseases have been increasing worldwide and reported in many animals including dogs and cats. Limited or no data are currently available regarding canine and feline vector-borne diseases in Saudi Arabia and limited information is available from other Middle Eastern countries. The aim of this study was to compare vector-borne disease prevalence between two bio-climatically distinct regions of Saudi Arabia, Riyadh province that is arid positioned at low elevation and Asir province that is humid at high elevation. Methods: Blood samples from 74d ogs from Riyadh province and 70 dogs and 44 cats from Asirprovince were collected and examined for the presence of genomic DNA of Babesia s pp, Anaplasma spp., Ehrlichia s pp., Bartonella spp., Mycoplasma spp., and Hepatozoon spp. by polymerase chain reaction (PCR), Multiplex - tandem PCR (MT-PCR) and Sanger sequencing. Results: Seventy four dogs were tested from Riyadh province and found be negative of any pathogen. Of the 70 dogs examined from Asir province 45(64.3%) were positive. Specifically, 40 (57.1%) dogs were positive for A.platy s, 20 (28.5%) for B.vogeli , 11(15.7%) for My.Haemocanis , two (2.85%) for Candidatus Mycoplasma haematoparvum and one (1.4%) for Br.henselae . Fourteen out of 44 cats (31.8%) were positive for one of the detected vector-borne pathogens. Six cats (13.6%) were positive for Candidatus Mycoplasma haemominutum and My.haemofelis, respectively, four cats (9.2%) were positive for Br.Henselae , two (4.54%) for Candidatus Mycoplasma haematoparvum and one (2.27%) for A. platy s. Conclusions: The results of this study report the occurrence of A. platys, B. vogeli, Br. henselae , and My. haemocanis in dogs and of A. platys , Br. henselae , My.haemofelis and Candidatus Mycoplasma haemominutum in cats from Asir province Further molecular investigations are strongly recommended in order to reduce the risk of dogs and cats acquiring vector-borne diseases in Saudi Arabia.


Introduction
Arthropods such as ticks, fleas and mosquitoes are globally important vectors of a wide range of viral, bacterial and protozoal pathogens resulting in a variety of animal diseases [1,2]. In some cases, vector-borne diseases are zoonotic and a direct threat to human health and animal welfare [3,4].
Other effects of arthropods feeding include anaemia, paralysis, immuno-suppression and invasion of tick bite wounds by secondary bacterial pathogens. Vector-borne diseases are often widespread in tropical and subtropical regions, including in the Middle East, due to optimal climatic conditions for vectors such as ticks, fleas and mosquitoes [5]. Tick infestations of dogs and cats are common in Saudi Arabia and mainly involves Hyalomma dromedarii (Koch, 1844) and Rhipicephalus sanguineus sensulato (s.l.)(Latreille, 1806) [6,7], which is a competentvector of diseases such as anaplasmosis, babesiosis, ehrlichiosis, hepatozoonosis and different Rickettsia species, among others [8].
The prevalence of vector-borne disease in a population closely reflects the distribution and density of such vectors [9,10]. Vector-borne disease in dogs and cats can be caused by rickettsial parasites (e.g. Anaplasma, Rickettsia) and haemoplasmas species (e.g. Mycoplasma haemofelis, Mycoplasma haematoparvum), resulting in potentially fatal and often persistent infections [10,11,12]. Studies in countries situated in and around the Middle Eastreport the presence of vector-borne pathogens including Anaplasma platys, Ehrlichia canis, Bartonella spp.,and Babesia spp. in dogs and cats [9,13,14,15,16,17,18,19].Currently, limited studies have examined the presence of vector-borne disease in dogs and cats in Saudi Arabia [7,20,21,22,23]. Therefore, the aim of this study was to evaluate the presence of vector-borne pathogens in dogs and cats in two different climatic zones of Saudi

Ethical approval
This study was revised and approved by the Ethical Research Committee, Department of Biological Science, Shaqra University, according to the ethical principles of human and animal research (Approval no. SH 06-2018).

Study areas
The investigation was conducted from November 2018 to August 2019 in two provinces of Saudi Arabia.The Riyadh province of Saudi Arabia has an area of 404,240 km² and is located in the central part of Saudi Arabia between 24˚.38˚N and 46˚.43˚E (Figure 1). This province is characterised by very hot summers with an average high temperature of 45˚C in July. Winters are cold, the overall climate is arid, receiving very little annual rainfall (21.4 mm), with the relative humidity ranging from 10% to 47% throughout the year. Riyadh province is also known to have many dust storms (http://www.pme.gov.sa). Asir province, has an area of 76,690 km² and is located in the southwestern part of Saudi Arabia between 19°0′N and 43°0′E (Figure 1). Asir province is situated on a high plateau that receives more rainfall than the rest of the country and contains the country's highest peaks, which rise to almost 3,000 m. Asir has a tropical and subtropical climate and the average annual rainfall in the highlands is expected torange from 300 to 500 ml across two rainy seasons. As a result, there is much more natural vegetation and forests (http://www.pme.gov.sa).

2.3.Sampling of dogs and cats and blood collections
Stray dogs and cats from the two provinces were trapped by a live bait traps (Havahart ® ) and

DNA extraction
Total genomic DNA (gDNA) was isolated from the blood samples using the Wizard® Genomic DNA Purification Kit (Promega, Madison,WI, USA) and eluted into 50 μl or 100 μl of elution buffer as per the manufacturer's instruction. An aliquot between 50 μl and 100 µl of gDNA from each of the samples was stored at −80°C prior to being sent to Veterinary Pathology Diagnostic Services (VPDS), Sydney School of Veterinary Science, The University of Sydney for PCR analysis. Upon arrival atVPDS gDNA was stored at -20 ˚C for up to 1 month prior to molecular diagnostics.

Commercial Multiplexed Tandem PCR (MT-PCR) for Small Animal Anaemia
A commercial diagnostic MT-PCR panel for small animal anaemia was performed using the mini-plex 12 system (R910738, AusDiagnostics Pty. Ltd., Australia) as per the manufacturers' instruction. The MT-PCR assay is a two-step nested PCR assay simultaneously targeting B. gibsoni, B. vogeli, M.
haematoparvum, M. Haemocanis and A. platys and was run on the Easy-Plex TM platform (AusDiagnostics Pty. Ltd., Australia). The assay was run in duplicate using 10 µlundiluted samples (n=188). Each run included controls to detect PCR inhibition (SPIKE) and sample adequacy control (ANONO) as per the manufacturers' protocol (AusDiagnostics Pty. Ltd., Australia).
The positive samples were tested using 16S rRNA primers (S0697/S0698) for Anaplasma platys [24], 18S rRNA primers (S0701/S0702) for the universal amplification of Mycoplasma spp. [25] and 18S rRNA primers for Babesia spp. [26]. All PCR reactions were run using MyTaq TM RedMix(Bioline, Australia) in a Veriti Thermal Cycler (Life Sciences, Australia). Primers were included at a final concentration of 400nM/µl and 2 µl of template DNA was used per reaction. All non-nested PCR were run using the following cycling conditions: 95 °C for 1 min and 35 cycles of 95 °C for 15 s, 50 °C for 15 s and 72 °C for 10 s followed by 72 °C for 5 min. PCR reactions with Babgen-F/Babgen-R primers were run with an annealing temperature of 55 °C, and all other conditions as previously described. The first round of the nested PCR was run using the following cycling conditions: 95 °C for 1 min and 35 cycles of 95 °C for 15 s, 55 °C for 15 s and 72 °C for 10 s followed by 72 °C for 5 min. The second round of the nested PCR was run using a 60 °C annealing temperature with all other conditions as described for the first round. A negative and positive control was included in all assays. All PCR products were separated by electrophoresis in 2% agarose gel stained with GelRed TM (Biotium, USA) and visualised using UV light. Discrete bands of expected size were submitted for bidirectional sequencing using amplification primers (Macrogen, South Korea). Sequences were assembled and compared to closely related sequences using CLC Main Workbench 6.8.1 (CLC bio, Denmark).

Real-time PCR: Bartonella spp.
A multiplex TaqMan probe real time PCR assay targeting the gltAgene of Rickettsia spp. was multiplexed with an assay targeting the ssrA gene of Bartonella spp. [27,28,29]. PCR reactions were run using SensiFAST TM Probe No-ROX Kit (Bioline, Australia) on a CFX95 Touch TM Real-Time PCR detection system (BioRad Laboratories Inc., Australia).

2.7.Statistical analysis
Statistical analyses were performed with the statistics package SPSS (v.17.0; IBM, New York, New York). Positive PCR test was set as an outcome variable and the independent variables were age, gender and, health status. The effect of independent variables on the outcome variables were evaluated by chi-square and Fisher's exact test and Odds Ratio (OR) calculation. Differences were considered significant if the P value was <0.05.
Most of the detected pathogens are vectored by Rhipicephalus sanguineus (s.l), a tick species displaying a worldwide geographical distribution [7,36]. Rh. sanguineus can infest a wide range of domestic and wild animals, including dogs, cats, rodents and birds [37]. Parasitism of Rh.
sanguineus on hosts other than dogs is quite unusual in several regions [38]. Rh. sanguineus has been reported in low prevalence in domestic and wild animals including dogs and cats fromcentral parts of Saudi Arabia [6,39,40], along with western and southern Saudi Arabia [41,42,43]. The climate of Saudi Arabia has shown to be potentially suitable to the perpetuation of vectors and transmission of several arthropod-borne diseases [44,45]. Indeed the environmental conditions of Saudi Arabia aresuitablefor the development of different tick species due to wide range of climatic conditions [46]. Climate conditions, animals diversity and vegetations vary and are different between Riyadh province and Asir province. Riyadh province is arid and positioned at low elevation, while Asir province is humid at a higher elevation. It has been shown that Rh. sanguineus can develop well under different conditions in terms of temperature (e.g., 20-35°C) and relative humidity (e.g., 35-95%) [47]. In a previous study Chandra et al., (2019) have shown that H. Dromedarii is the most common tick parasitising dogs in Riyadh province.This tick preferentially parasitises camels, although has been known to parasitise other ungulates [48,49]. In this study, we did not collected ticks from dogs and cats from Asir province hence it is difficult to conclude the presence of these parasites in Asir province but not in Riyadh province.

Conclusion
This study expands existing information on the distribution of canine and feline vector-borne diseases

Disclosure
The authors declare that they have no competing interests.

Funding
Not applicable.

Consent for publication
Not applicable.

Ethics approval and consent to participate
Blood sampling for this study was approved by the Ethical Research Committee, Shaqra University and complied with relevant guidelines for animal handling and welfare (Approval no. SH 06-2018)

Authors' contributions
ADA and JŠ participated in the study design. MSA coordinated, ASA,IOA and MAY collected ticks and blood samples and performed blood DNA isolation. NEDCand JŠ performed MT-PCR, PCR and qPCR.
ADA, NEDC and JŠ interpreted the PCR results. ADA and JŠ wrote the manuscript. All authors read and approved the final manuscript. Figure 1 Map showing the study sites of Riyadh province and Asir province, Saudi Arabia. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.