The world-famous markets of Marrakech, known as souks, represent a unique setting where reptiles are sold for medicinal, magical, or pet purposes, and used for snake charming. Souk is an Arabic term which means “the market”, and in North Africa and the Middle East it is an important center, not only of commerce but of tradition, and culture of Arab-Islamic societies [1]. The origin of these important markets is believed to be strictly associated with the evolution and diffusion of the Islamic societies. Nonetheless, archeological documentation of ancient souks is scarce, yet records of souks date back from 3000 B.C. from Anatolian Persia [2].
In Morocco, the souks of Marrakech represent the largest and most famous markets, becoming in recent years not only the heart of Marrakech’s commerce, trade, and art, but also an important touristic hotspot [3]. Marrakech’s old city or medina concentrates the largest number of souks, which are near the enigmatic Jemaa-El-Fna square (also known as Jamâ-El-Fna or Djemaa-El-Fna square; literally: crossroads of the arts). This enigmatic square was founded in the 11th century, and since then, it is a space of great complexity, involving Moroccan traditions, displayed through art, religion, music and gastronomy. Given its souks and its cultural heritage which has given the city the designation of World Heritage Site by UNESCO twice, Marrakech has become one of the most known tourist destinations in Morocco and North Africa [4].
Within Jemaa-El-Fna square, presence of monkeys and snakes is common [3]. Certainly, despite the charm and touristic attractiveness of the Souks, there is the risk of exploitation of wild animals [5,6]. Specifically, regarding reptiles’ presence in the souks of Marrakech, their use is associated to medicinal purposes or snake charming [6,7]. Indeed, the souks within the medina of Marrakech are full of live reptiles, mainly spur-thighed tortoises (Testudo graeca), Mediterranean chameleons (Chamaeleo chamaeleon), and occasionally Bell’s Dabb monitor lizards (Uromastyx acanthinura) and desert lizards (Varanus griseus), that are used with etnoherpetological purposes such as traditional medicine and magic [7].
Likewise, animal anatomical pieces are also commercialized with the same purposes being the desert lizard, Nile crocodile (Crocodylus niloticus), and the African rock python (Python sebae), both species being exotic to Morocco, as well as heads of the Egyptian cobra (Naja haje), present throughout the souks of Marrakech [6]. On the other hand, snake charming is still a prevalent craft in large cities of Morocco, being Marrakech the main center of this activity in the country, with descriptions of its existence since the late 1700s [8]. Snake charming is mainly practiced by a religious brotherhood called Aissawa, which claim to be immune to the snakes’ venom.
Commonly used species for snake charming in the Jemaa-El-Fna square are the Egyptian cobra and the puff adder (Bitis arietans), followed by the horned viper (Cerastes cerates) and Moorish viper (Daboia mauritanica). Mildly to non-venomous species are also used such as the Montpellier snake (Malpolon monspessulanus), and to a lesser extent the horseshoe whip snake (Hemorrhois hippocrepis) [8]. Snakes used by charmers are generally captured (from April to October) and brought to the souks of Marrakech from the Atlantic belt of South-Western Morocco, where species of snakes (more than 27 species) thrive [8].
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This unique epidemiological context, where reptiles are in constant contact with humans, has led to concerns about the transmission of zoonotic pathogens. The overall welfare, husbandry and living conditions of animals within the souks are scarce. These unhygienic conditions, coupled with the constant handling and proximity to people, are driving factors for zoonotic pathogens’ transmission. Indeed, in this context, humans may be exposed to reptile-borne pathogens and reptile vector-borne diseases (RBVDs), given their proximity, and constant interaction [9,10]. Within the various transmission pathways, environmental contamination or oral-fecal transmitted pathogens have greater possibilities to infect people via reptile handling. Besides Salmonella, many other bacteria and parasites can be transmitted [9,11]. However, there are no studies assessing the prevalence of these pathogens associated with the human-reptile interface.
Moreover, other zoonotic pathogens associated to reptiles (e.g., Anaplasma, Borrelia, Rickettsia) are transmitted by vectors (i.e., mosquitoes, ticks, and sand flies) [10]. Previous studies in Moroccan herpetofauna have addressed Salmonella [12], and ticks [13] from tortoises, as well as herpetophilic sand flies’ species and their potential role in the transmission of leishmaniases [14]. In addition, ecological studies have been performed on the prevalence and distribution of hemoparasites, such as Hepatozoon spp. associated to Moroccan reptiles [15,16], or on the prevalence of parasitic fauna of endemic species of reptiles [17].
Thus, a study was conducted to identify the parasites and pathogens present in blood and feces associated with handled reptiles in the markets of Marrakech to assess the risk of zoonotic transmission within the reptile-human interface.
Materials and Methods
The study was conducted in accordance with all applicable international, national, and/or institutional guidelines for the care and use of animals. In October 2022, reptiles kept within the proximities of Jeema-El-Fna square of Marrakech (Fig 1) were examined, morphologically identified to species level using reference keys or checklists [18-20], and sampled after authorization of their owners (i.e., vendors or snake charmers; Fig 1).
A blood sample (Fig 2) was obtained from each animal. Blood (~100 μl to 1ml) was drawn from snakes and lizards using the ventral coccygeal vein, whereas tortoises’ blood was drawn from the subcarapacial sinus. Blood was divided in Whatman FTA Cards and 1.5 ml Eppendorf tubes which were later stored at -20 °C. Cloacal swabs were performed from all animals and stored at -20 °C. Blood smears were performed from all animals and then assessed for the presence of hemoparasites [21] using Diff-Quik stain [22].
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DNA was extracted from individual blood samples (n = 112) and cloacal swabs (n = 102) using a commercial kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany), according to the manufacturer’s instructions and analyzed for the detection of different microrganisms and parasites (see below). Details regarding cPCR and qPCR protocols are reported in Table 1. All cPCR products were examined on 2% agarose gel stained with GelRed (VWR International PBI, Milan, Italy) and visualized on a GelLogic 100 gel documentation system (Kodak, New York, USA).
Amplicons were then purified and sequenced in both directions using the same primers as for PCRs, by the Big Dye Terminator version 3.1 chemistry in a 3130 Genetic Analyzer (Applied Bio-systems, Foster City, CA, USA). Nested PCRs were performed for Giardia spp. and Cryptosporidium spp. as follows. For Giardia, a nested PCR amplifying partial triosephosphate isomerase (tpi) gene (532 bp) was used, to also detect all known assemblages [24,25].
Rickettsial gltA as well as 16S rRNA sequences from Anaplasma spp. were separately aligned against those closely related species available from GenBank database using the ClustalW application within MEGA7 software [37]. The Akaike Information Criterion (AIC) option in MEGA7 was used to establish the best nucleotide substitution model adapted to each sequence alignment. Tamura-Nei model with a invariant sites (I) [38] was used to generate the gltA and the 16S rRNA trees. A maximum likelihood (ML) phylogenetic inference was used with 2000 bootstrap replicates to generate the phylogenetic tree in MEGA7.
Results
Overall, 118 reptile specimens were examined and screened represented by two orders [Squamata (Amphisbaenia with one species, Sauria with two species in two families, Ophidia with five species in three families), and Testudines (one species); Table 2. Species of reptiles commercialized in the markets were from Amphisbaenia, Sauria and Testudines (Fig 3). In addition, 68 snake specimens used by charmers were screened represented mainly by Montpellier snakes, followed by puff adders, Egyptian cobras, eastern Montpellier snake, and one individual of horseshoe whip snake (Table 2). Two of the before mentioned species are considered highly venomous (i.e., puff adder, Egyptian cobra), whereas both species of Malpolon are considered mildly venomous rear-fanged (i.e., opisthoglyphous) snakes.
Positive blood smear observation yielded the presence of hemogregarines’ gametocytes in 22% of examined individuals (26/118; Fig 5). Gamonts were only observed in the five species of snakes (Table 3). Overall, 28.9% (34/118) of examined reptiles were positive to at least one molecularly identified pathogen, being 9.3% (11/118) animals owned by vendors and 19.5% (23/118) snakes used by charmers. Molecular screening of pathogens in blood rendered positive results for Anaplasma/Ehrlichia spp., Rickettsia spp. (gltA), Babesia/Theileria spp., and Leishmania spp. (ITS) (Table 3), whereas all samples were negative for Borrelia burgdorferi sensu lato, Spotted Fever Group Rickettsiae or Coxiella burnetii.
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Specifically, four snakes (3.4%) were positive for Anaplasma spp., with two Montpellier snakes positive to Anaplasma phagocytophilum (i.e., 99.7% nucleotide identity with MT126499) of horses from Turkey. All generated sequences of Anaplasma sp. clustered within the species of Anaplasma (A. phagocytophilum, Anaplasma platys, Anaplasma odocoilei) with high bootstraps values (i.e., 87%) (Fig 6A). On the other hand, 16S rRNA target gene also amplified in a puff adder for the endosymbiont Candidatus Midichloria mitochondrii (100.00% nucleotide identity with EU780455 of Cimex lectularius). In addition, 4.2% of reptiles (5/118; four puff adders and one Mediterranean chameleon) were positive for Rickettsia spp. The four puff adders were positive to Rickettsia asiatica (97.7% homology with AP019563), and the Mediterranean chameleon to Candidatus Rickettsia asembonensis (100% nucleotide identity with MK923743 of Ctenocephalides canis).
Phylogenetic inference clustered four sequences of Rickettsia sp. from puff adders with Rickettsia helvetica, whereas Rickettsia sp. sequence from a Mediterranean chameleon clustered with R. asembonensis, Rickettsia felis and Candidatus Rickettsia senegalensis (Fig 6B). Furthermore, despite the high prevalence in blood smears, only three sequences were obtained for Hepatozoon spp. in a Mediterranean chameleon, a Montpellier snake, and a puff adder (99.9% nucleotide identity with KC696565), previously detected in Psammophis schokari snake form North Africa (Table 3).
Conversely, molecular screening of pathogens in feces revealed positive results for a great variety of agents. Particularly, a myriad of sequences were obtained with the cox1 gene, from nematodes and cestodes, to fungi and bacteria. Nematodes were detected in eight (6.7%) spur-thighed tortoises and two (1.7%) Montpellier snakes. Sequences generated were similar (83 to 89%) to Enterobius (HQ317434), Syphacia (MH427272) and Trypanoxyuris (KJ939328) genera. Moreover, cestodes sequences were retrieved from two snakes (eastern Montpellier snake and Montpellier snake) represented by Mesocestoides (MH463505) and Penetrocephalus (KR780799) genera (83% of nucleotide identity). Lastly, Cryptosporidium spp. 18S rRNA gene nPCR revealed positive samples for Cryptosporidium sp. apodemus genotype I (MH912997; 92% of nucleotide identity) in one Moroccan worm lizard. Also, the nPCR yielded positive results for bacteria such as Pseudomonas aeruginosa in an Egyptian cobra, and Morganella morganii from a puff adder.
The detection of these pathogens highlights the potential for zoonotic transmission in the unique environment of Marrakech's souks, where humans and reptiles interact closely.
| Reptile Species | Pathogen | Reference |
|---|---|---|
| Puff Adder | Hepatozoon sp. | KC696565 |
| Moroccan Worm Lizard | Cryptosporidium sp. | MH912997 |
| Montpellier Snake | Hepatozoon sp. | KC696565 |
| Montpellier Snake | Anaplasma sp. | MK041546 |
| Montpellier Snake | Anaplasma sp. | MW790941 |
| Mediterranean Chameleon | Hepatozoon sp. | KC696565 |
| Eastern Montpellier Snake | Mesocestoides sp. | MH463505 |
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