Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

BACKGROUND: Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. RESULTS: Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. CONCLUSIONS: P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Molecular Detection of Candidatus Orientia chuto in Wildlife, Saudi Arabia.

Scrub typhus is a zoonosis caused by 3 species of Orientia bacteria, including Candidatus Orientia chuto. This species is known only from a human case in Dubai and infections in wildlife in Kenya. We report molecular detection of Candidatus O. chuto in 2 wild rodent species from Saudi Arabia.

Middle East Respiratory Syndrome Coronavirus Seropositivity in Camel Handlers and Their Families, Pakistan.

A high percentage of camel handlers in Saudi Arabia are seropositive for Middle East respiratory syndrome coronavirus. We found that 12/100 camel handlers and their family members in Pakistan, a country with extensive camel MERS-CoV infection, were seropositive, indicating that MERS-CoV infection of these populations extends beyond the Arabian Peninsula.

New insight into genetic variation and haplotype diversity of Fasciola hepatica from Algeria.

The liver fluke Fasciola hepatica is the main cause of fasciolosis in North Africa leading to significant economic losses and public health problems. In this study, the ribosomal internal transcribed spacer (ITS), cytochrome c oxidase I (COI), the mitochondrial region spanning the COI-trnT-rrnL, and the NADH dehydrogenase subunit I (NADI) markers were used to characterize Fasciola flukes from Algeria. Fasciola appeared widespread from the east to the west of Algeria. Among 1701 sampled cattle from 8 Algerian provinces, 5% were infected. Using morphological and morphometric analysis, one morphotype of Fasciola was observed. Nuclear ITS marker indicated that all collected flukes belong to F. hepatica. Multiple alignments of ITS dataset revealed two haplotypes, one described here for the first time. Analysis of molecular variance (AMOVA) of mitochondrial markers revealed weak population structure in Algeria. Mismatch distributions, neutrality tests, and median-joining network analysis all were compatible with a recent expansion of Algerian F. hepatica population. Fasciolosis appeared common in Algerian cattle, it seems that the absence of control strategy coupled to the favorable Mediterranean climate may lead to a reconstruction and dispersion of its populations. This study provides important results concerning the genetic characterization and variability of F. hepatica in Algeria as well as the significant role of cattle importation in shaping its dispersal route worldwide.

Infectious diseases epidemic threats and mass gatherings: refocusing global attention on the continuing spread of the Middle East Respiratory syndrome coronavirus (MERS-CoV).

Media and World Health Organization (WHO) attention on Zika virus transmission at the 2016 Rio Olympic Games and the 2015 Ebola virus outbreak in West Africa diverted the attention of global public health authorities from other lethal infectious diseases with epidemic potential. Mass gatherings such as the annual Hajj pilgrimage hosted by Kingdom of Saudi Arabia attract huge crowds from all continents, creating high-risk conditions for the rapid global spread of infectious diseases. The highly lethal Middle Eastern respiratory syndrome coronavirus (MERS-CoV) remains in the WHO list of top emerging diseases likely to cause major epidemics. The 2015 MERS-CoV outbreak in South Korea, in which 184 MERS cases including 33 deaths occurred in 2 months, that was imported from the Middle East by a South Korean businessman was a wake-up call for the global community to refocus attention on MERS-CoV and other emerging and re-emerging infectious diseases with epidemic potential. The international donor community and Middle Eastern countries should make available resources for, and make a serious commitment to, taking forward a "One Health" global network for proactive surveillance, rapid detection, and prevention of MERS-CoV and other epidemic infectious diseases threats.

On the origin of mongrels: evolutionary history of free-breeding dogs in Eurasia.

Although a large part of the global domestic dog population is free-ranging and free-breeding, knowledge of genetic diversity in these free-breeding dogs (FBDs) and their ancestry relations to pure-breed dogs is limited, and the indigenous status of FBDs in Asia is still uncertain. We analyse genome-wide SNP variability of FBDs across Eurasia, and show that they display weak genetic structure and are genetically distinct from pure-breed dogs rather than constituting an admixture of breeds. Our results suggest that modern European breeds originated locally from European FBDs. East Asian and Arctic breeds show closest affinity to East Asian FBDs, and they both represent the earliest branching lineages in the phylogeny of extant Eurasian dogs. Our biogeographic reconstruction of ancestral distributions indicates a gradual westward expansion of East Asian indigenous dogs to the Middle East and Europe through Central and West Asia, providing evidence for a major expansion that shaped the patterns of genetic differentiation in modern dogs. This expansion was probably secondary and could have led to the replacement of earlier resident populations in Western Eurasia. This could explain why earlier studies based on modern DNA suggest East Asia as the region of dog origin, while ancient DNA and archaeological data point to Western Eurasia.

Out of Africa, but how and when? The case of hamadryas baboons (Papio hamadryas).

Many species of Arabian mammals are considered to be of Afrotropical origin and for most of them the Red Sea has constituted an obstacle for dispersal since the Miocene-Pliocene transition. There are two possible routes, the 'northern' and the 'southern', for terrestrial mammals (including humans) to move between Africa and Arabia. The 'northern route', crossing the Sinai Peninsula, is confirmed for several taxa by an extensive fossil record, especially from northern Egypt and the Levant, whereas the 'southern route', across the Bab-el-Mandab Strait, which links the Red Sea with the Gulf of Aden, is more controversial, although post-Pliocene terrestrial crossings of the Red Sea might have been possible during glacial maxima when sea levels were low. Hamadryas baboons (Papio hamadryas) are the only baboon taxon to disperse out of Africa and still inhabit Arabia. In this study, we investigate the origin of Arabian hamadryas baboons using mitochondrial sequence data from 294 samples collected in Arabia and Northeast Africa. Through the analysis of the geographic distribution of genetic diversity, the timing of population expansions, and divergence time estimates combined with palaeoecological data, we test: (i) if Arabian and African hamadryas baboons are genetically distinct; (ii) if Arabian baboons exhibit population substructure; and (iii) when, and via which route, baboons colonized Arabia. Our results suggest that hamadryas baboons colonized Arabia during the Late Pleistocene (130-12 kya [thousands of years ago]) and also moved back to Africa. We reject the hypothesis that hamadryas baboons were introduced to Arabia by humans, because the initial colonization considerably predates the earliest records of human seafaring in this region. Our results strongly suggest that the 'southern route' from Africa to Arabia could have been used by hamadryas baboons during the same time period as proposed for modern humans.

Where Do Core Thalamocortical Axons Terminate in Mammalian Neocortex When There Is No Cytoarchitecturally Distinct Layer 4?

Although the mammalian cerebral cortex is most often described as a hexalaminar structure, there are cortical areas (primary motor cortex) and species (elephants, cetaceans, and hippopotami), where a cytoarchitecturally indistinct, or absent, layer 4 is noted. Thalamocortical projections from the core, or first order, thalamic system terminate primarily in layers 4/inner 3. We explored the termination sites of core thalamocortical projections in cortical areas and in species where there is no cytoarchitecturally distinct layer 4 using the immunolocalization of vesicular glutamate transporter 2, a known marker of core thalamocortical axon terminals, in 31 mammal species spanning the eutherian radiation. Several variations from the canonical cortical column outline of layer 4 and core thalamocortical inputs were noted. In shrews/microchiropterans, layer 4 was present, but many core thalamocortical projections terminated in layer 1 in addition to layers 4 and inner 3. In primate primary visual cortex, the sublaminated layer 4 was associated with a specialized core thalamocortical projection pattern. In primate primary motor cortex, no cytoarchitecturally distinct layer 4 was evident and the core thalamocortical projections terminated throughout layer 3. In the African elephant, cetaceans, and river hippopotamus, no cytoarchitecturally distinct layer 4 was observed and core thalamocortical projections terminated primarily in inner layer 3 and less densely in outer layer 3. These findings are contextualized in terms of cortical processing, perception, and the evolutionary trajectory leading to an indistinct or absent cortical layer 4.

Mobilisation of jerboa kidney gene networks during dehydration and opportunistic rehydration.

Desert animals have evolved systems that enable them to thrive under dry conditions. Focusing on the kidney, we have investigated the transcriptomic adaptations that enable a desert rodent, the Lesser Egyptian Jerboa (Jaculus jaculus), to withstand water deprivation and opportunistic rehydration. Analysis of the whole kidney transcriptome showed many differentially expressed genes in the Jerboa kidney, 6.4% of genes following dehydration and an even greater number (36.2%) following rehydration compared to control. Genes correlated with the rehydration condition included many ribosomal protein coding genes suggesting a concerted effort to accelerate protein synthesis when water is made available. We identify an increase in TGF-beta signaling antagonists in dehydration (e.g., GREM2). We also describe expression of multiple aquaporin and solute carrier transporters mapped to specific nephron segments. The desert adapted renal transcriptome presented here is a valuable resource to expand our understanding of osmoregulation beyond that derived from model organisms.

Examination of head versus body heading may help clarify the extent to which animal movement pathways are structured by environmental cues?

Understanding the processes that determine how animals allocate time to space is a major challenge, although it is acknowledged that summed animal movement pathways over time must define space-time use. The critical question is then, what processes structure these pathways? Following the idea that turns within pathways might be based on environmentally determined decisions, we equipped Arabian oryx with head- and body-mounted tags to determine how they orientated their heads - which we posit is indicative of them assessing the environment - in relation to their movement paths, to investigate the role of environment scanning in path tortuosity. After simulating predators to verify that oryx look directly at objects of interest, we recorded that, during routine movement, > 60% of all turns in the animals' paths, before being executed, were preceded by a change in head heading that was not immediately mirrored by the body heading: The path turn angle (as indicated by the body heading) correlated with a prior change in head heading (with head heading being mirrored by subsequent turns in the path) twenty-one times more than when path turns occurred due to the animals adopting a body heading that went in the opposite direction to the change in head heading. Although we could not determine what the objects of interest were, and therefore the proposed reasons for turning, we suggest that this reflects the use of cephalic senses to detect advantageous environmental features (e.g. food) or to detect detrimental features (e.g. predators). The results of our pilot study suggest how turns might emerge in animal pathways and we propose that examination of points of inflection in highly resolved animal paths could represent decisions in landscapes and their examination could enhance our understanding of how animal pathways are structured.

DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Redescription of Eimeria dorcadis Mantovani, 1966 (Apicomplexa: Eimeriidae) from the dorcas gazelle (Gazella dorcas) in Saudi Arabia.

Eimeria dorcadis Mantovani, 1966 is redescribed from dorcas gazelle (Gazella dorcas (L.)) from Saudi Arabia. Oocysts were detected in 7 out of 22 faecal samples (32%) using floatation method. The sporulated oocysts are cylindrical, slightly flattened at the micropylar pole, measure in average 32 x 19 microm (27-36 x 16-24 microm), length/width ratio being 1.7 (1.5-2.1). Oocyst wall is 1.2 microm thick, smooth, double-layered; outer layer is slightly thicker, light blue in colour; inner layer brownish, with micropyle in the inner layer and apparently continual outer one, measures 2.2 microm, but lacks a micropylar cap. The sporocyst elongate-ellipsoidal, measures 14 x 8 microm (12-17 x 6-9 microm), length/width ratio being 1.8, with sporocyst residuum as circular compact, coarse, refractile granules. Stieda body is present, while substieda body is absent. Sporozoites banana-shaped, measure 11 x 2.5 microm, each with a large spheroidal refractile body at the wider pole. Sporulation time is 2-3 days at 25 +/- 2 degrees C.

Genetic diversity of wild rodents and detection of Coxiella burnetii, the causative agent of Q fever, in Saudi Arabia.

Throughout history, wildlife has been regarded as a major source of infectious diseases. Rodentia, the most speciose order of mammals, whose members are recognised hosts of more than 60 zoonotic diseases, represent a potential threat to human health. Recently, epidemiological data from Saudi Arabia indicated an actual growth in the number of emerging and/or re-emerging cases of several zoonoses. However, there is a lack of studies focusing on the molecular taxonomy of rodents and the pathogens they may harbour in this region. In this study, the first molecular characterisation of six rodent taxa in this region is provided, based on partial Cyt B and 16S genes. The data confirm the spread of rodent-associated C. burnetii strains in Jazan, southwestern Saudi Arabia. The PCR targeting IS111, the multi-copy transposase gene, revealed 17.5% (36/205) positive samples, whereas the second nested PCR, targeting the single-copy Com1 gene, revealed 16.6% (34/205) positive samples. Phylogenetic and network analyses indicated the presence of four haplotypes of C. burnetii within the studied localities. One major haplotype (H-2) was observed in all rodent species and from 18 localities. The infection rates of C. burnetii among studied species, localities and habitats were not significantly different (>0.05). Our results facilitate the assessment of the health risk associated with rodents and the development of strategies to control the increasing impacts of Q fever.

Automatic barcode gap discovery reveals diverse clades of Rhipicephalus spp. and Haemaphysalis spp. ticks from small mammals in 'Asir, Saudi Arabia.

BACKGROUND: The ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula. METHODS: In this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1, 16S rRNA and 12S rRNA. RESULTS: We obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog (Paraechinus aethiopicus). Morphologically, the Rhipicephalus ticks resembled R. camicasi, but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis, R. guilhoni and R. linnaei. The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species. CONCLUSIONS: This apparent high level of tick diversity observed in a single study site of only ~ 170 km2, on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula.

Amplification of potential thermogenetic mechanisms in cetacean brains compared to artiodactyl brains.

To elucidate factors underlying the evolution of large brains in cetaceans, we examined 16 brains from 14 cetartiodactyl species, with immunohistochemical techniques, for evidence of non-shivering thermogenesis. We show that, in comparison to the 11 artiodactyl brains studied (from 11 species), the 5 cetacean brains (from 3 species), exhibit an expanded expression of uncoupling protein 1 (UCP1, UCPs being mitochondrial inner membrane proteins that dissipate the proton gradient to generate heat) in cortical neurons, immunolocalization of UCP4 within a substantial proportion of glia throughout the brain, and an increased density of noradrenergic axonal boutons (noradrenaline functioning to control concentrations of and activate UCPs). Thus, cetacean brains studied possess multiple characteristics indicative of intensified thermogenetic functionality that can be related to their current and historical obligatory aquatic niche. These findings necessitate reassessment of our concepts regarding the reasons for large brain evolution and associated functional capacities in cetaceans.

Human-modified canids in human-modified landscapes: The evolutionary consequences of hybridization for grey wolves and free-ranging domestic dogs.

Introgressive hybridization between domestic animals and their wild relatives is an indirect form of human-induced evolution, altering gene pools and phenotypic traits of wild and domestic populations. Although this process is well documented in many taxa, its evolutionary consequences are poorly understood. In this study, we assess introgression patterns in admixed populations of Eurasian wolves and free-ranging domestic dogs (FRDs), identifying chromosomal regions with significantly overrepresented hybrid ancestry and assessing whether genes located within these regions show signatures of selection. Although the dog admixture proportion in West Eurasian wolves (2.7%) was greater than the wolf admixture proportion in FRDs (0.75%), the number and average length of chromosomal blocks showing significant overrepresentation of hybrid ancestry were smaller in wolves than FRDs. In wolves, 6% of genes located within these blocks showed signatures of positive selection compared to 23% in FRDs. We found that introgression from wolves may provide a considerable adaptive advantage to FRDs, counterbalancing some of the negative effects of domestication, which can include reduced genetic diversity and excessive tameness. In wolves, introgression from FRDs is mostly driven by drift, with a small number of positively selected genes associated with brain function and behaviour. The predominance of drift may be the consequence of small effective size of wolf populations, which reduces efficiency of selection for weakly advantageous or against weakly disadvantageous introgressed variants. Small wolf population sizes result largely from human-induced habitat loss and hunting, thus linking introgression rates to anthropogenic processes. Our results imply that maintenance of large population sizes should be an important element of wolf management strategies aimed at reducing introgression rates of dog-derived variants.

Multilocus approach reveals discordant molecular markers and corridors for gene flow between North African populations of Fasciola hepatica.

Fasciolosis is a foodborne trematodosis characterised by a worldwide distribution. Various approaches have been developed for the study of the causative agents of this parasitic infection: Fasciola hepatica, Fasciola gigantica and the aspermic intermediated forms (hybrid and introgressed). In the present study, novel and common molecular markers (pepck and pold, ITS, CO1, ND1 and CO1-trnT-rrnL) were used to characterise Fasciola flukes from the Tunisian-Algerian border, to estimate the gene flow between these populations and to evaluate the reliability of different molecular markers. All nuclear and mitochondrial markers, apart from pepck, supported the monophyly of the studied flukes identified as F. hepatica. Multiplex PCR for pepck revealed three different genotypes corresponding to F. hepatica (pepck-Fh), F. gigantica (pepck-Fg) and the aspermic Fasciola flukes (pepck-Fh/Fg). Sequence analysis of pepck revealed high polymorphism, length variation, within this intronic marker. The observed inconsistencies were due to the position of the forward primer within the intronic region. Pepck sequences showed different level of heterozygosity and homozygosity with length polymorphisms in the introns. Pepck multiplex PCR patterns could not differentiate between Fasciola species. All studies based on only pepck multiplex PCR with mitochondrial markers should be revised. Nuclear and mitochondrial markers revealed an important gene flow between Tunisian and Algerian populations of F. hepatica. The combination of nuclear and mitochondrial sequence analysis is still the best method to distinguish these taxa. Effective measures are needed in order to better control cross-country illegal trade of vector.

Genotyping of Clostridium perfringens Isolates from Domestic Livestock in Saudi Arabia.

The present study was undertaken to confirm the genetic identity of Clostridium perfringens isolates from domestic livestock in Saudi Arabia and to characterize the genes encoding to alpha, beta, epsilon, and iota (α-, ß-, ε-, and ι-) toxins. C. perfringens were confirmed in 104 out of 136 isolates on multiplex polymerase chain reaction using specific primers amplifying genes related to toxins produced by C. perfringens. Genes encoding α-toxins were detected in 104 samples. Of the isolates, 80.8% were diagnosed as type A, 15.4% as type D, 2.9% as type C, and 0.96% as type B. None of the isolates has genes encoding iota (ι-) toxin. All isolates investigated yielded enterotoxin (cpe) products and none yielded ß2 (cpb2-toxin) or NetB products. PLC gene sequences encoding α-toxin showed >96.7% similarity. Isolates which had α-toxins as well as enterotoxin (cpe) are regarded as type F. Phylogenetic analysis using maximum likelihood analysis yielded two clades, and the majority of the isolates were in one group while only two isolates clustered on the second clade. Within the Kingdom of Saudi Arabia strains, 54 variable positions and 23 polymorphic amino acids were noticed. Isolates with ε- and ß-toxins were variable and were found to be close to those published for C. perfringens. ETX gene sequences encoding ε-toxins were found to be related to CPE sequences.

Cross-sectional prevalence study of MERS-CoV in local and imported dromedary camels in Saudi Arabia, 2016-2018.

The Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) is an endemic virus in dromedaries. Annually, Saudi Arabia imports thousands of camels from the Horn of Africa, yet the epidemiology of MERS-CoV in these animals is largely unknown. Here, MERS-CoV prevalence was compared in imported African camels and their local counterparts. A total of 1399 paired sera and nasal swabs were collected from camels between 2016 and 2018. Imported animals from Sudan (n = 829) and Djibouti (n = 328) were sampled on incoming ships at Jeddah Islamic seaport before unloading, and local camels were sampled from Jeddah (n = 242). Samples were screened for neutralizing antibodies (nAbs) and MERS-CoV viral RNA. The overall seroprevalence was 92.7% and RNA detection rate was 17.2%. Imported camels had higher seroprevalence compared to resident herds (93.8% vs 87.6%, p <0.01) in contrast to RNA detection (13.3% vs 35.5%, p <0.0001). Seroprevalence significantly increased with age (p<0.0001) and viral RNA detection rate was ~2-folds higher in camels <2-year-old compared to older animals. RNA detection was higher in males verses females (24.3% vs 12.6%, p<0.0001) but seroprevalence was similar. Concurrent positivity for viral RNA and nAbs was found in >87% of the RNA positive animals, increased with age and was sex-dependent. Importantly, reduced viral RNA load was positively correlated with nAb titers. Our data confirm the widespread of MERS-CoV in imported and domestic camels in Saudi Arabia and highlight the need for continuous active surveillance and better prevention measures. Further studies are also warranted to understand camels correlates of protection for proper vaccine development.

Waterpipe smoking as a public health risk: Potential risk for transmission of MERS-CoV.

The Middle East Respiratory Syndrome (MERS-CoV) emerged in the Kingdom of Saudi Arabia in 2012 causing a critical challenge to public health. The epidemiology of MERS-CoV remain enigmatic as human-to-human transmission is not fully understood. One possible scenario that might play a role in the virus transmission is the cultural waterpipe smoking. Cafés providing waterpipe smoking in cities within Saudi Arabia have been moved to areas outside city limits that frequently place them close to camels markets. We report results of a surveillance study wherein waterpipe hoses throughout several regions in Saudi Arabia were tested for the presence of MERS-CoV. A total of 2489 waterpipe samples were collected from cities where MERS-CoV cases were continuously recorded. MERS-CoV RNA wasn't detected in collected samples. Irrespective of the negative results of our survey, the public health risk of waterpipe smoking should not be underestimated. To avoid a possible transmission within country where MERS-CoV is prevalent, we recommend the replacement of resusable hoses with "one-time-use" hoses in addition to a close inspection of waterpipe components to assure the appropriate cleaning and sanitization.