(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Parasites and microorganisms associated with the snakes collected for the “festa Dei serpari” in Cocullo, Italy [1]

['Jairo Alfonso Mendoza-Roldan', 'Department Of Veterinary Medicine', 'University Of Bari', 'Valenzano', 'Livia Perles', 'Ernesto Filippi', 'Biologist Consultant For The Cocullo Municipality', 'Rome', 'Nicole Szafranski', 'College Of Veterinary Medicine']

Date: 2024-03

Abstract While in much of the Western world snakes are feared, in the small, rural, mountainous town of Cocullo, in the middle of central Italy, snakes are annually collected and celebrated in a sacro-profane ritual. Every 1st of May, Serpari (snake catchers) capture and showcase dozens of non-venomous snakes to celebrate the ritual of San Domenico. In order to detect potential zoonotic pathogens within this unique epidemiological context, parasites and microorganisms of snakes harvested for the “festa dei serpari” ritual was investigated. Snakes (n = 112) were examined and ectoparasites collected, as well as blood and feces sampled. Ectoparasites were identified morpho-molecularly, and coprological examination conducted through direct smear and flotation. Molecular screenings were performed to identify parasites and microorganisms in collected samples (i.e., Mesostigmata mites, Anaplasma/Ehrlichia spp., Rickettsia spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Babesia/Theileria spp., Cryptosporidium spp., Giardia spp., Leishmania spp. and helminths). Overall, 28.5% (32/112) of snakes were molecularly positive for at least one parasite and/or microorganism. Endosymbiont Wolbachia bacteria were identified from Macronyssidae mites and zoonotic vector-borne bacteria (e.g., Rickettsia, Leishmania), as well as orally transmitted pathogens (i.e., Cryptosporidium, Giardia, Proteus vulgaris, Pseudomonas), were detected from blood and feces. Thus, given the central role of the snakes in the tradition of Cocullo, surveys of their parasitic fauna and associated zoonotic pathogens may aid to generate conservation policies to benefit the human-snake interactions, whilst preserving the cultural patrimony of this event.

Author summary The “festa dei serpari” is a unique sacro-profane ritual held each 1st of May in the small town of Cocullo, Central Italy. In this ceremony, dozens of non-venomous free-ranging snakes are captured by serpari (snake catchers) and showcased to thousands of pilgrims and tourists. Therefore, we aimed to assess the parasites and microorganisms of snakes within this unique epidemiological context to identify potential zoonotic pathogens. Snakes were examined and ectoparasites, blood and feces were collected, and morpho-molecular studies were performed. Overall, 28.5% (32/112) of snakes were positive for at least one parasite and/or microorganism. We identified new records of Mesostigmata mites as well as Leishmania tarentolae for the first time in Italian snakes. Importantly, we detected zoonotic microorganisms such as Rickettsia sp. in Aesculapian snake, as well as orally transmitted pathogens (i.e., Cryptosporidium, Giardia, Pseudomonas sp., Proteus vulgaris) from blood and feces of four species of snakes. Thus, snakes handled in this tradition may play a role in the zoonotic transmission of pathogens, given the contact with humans during this unique event.

Citation: Mendoza-Roldan JA, Perles L, Filippi E, Szafranski N, Montinaro G, Carbonara M, et al. (2024) Parasites and microorganisms associated with the snakes collected for the “festa Dei serpari” in Cocullo, Italy. PLoS Negl Trop Dis 18(2): e0011973. https://doi.org/10.1371/journal.pntd.0011973 Editor: Marcelo Larami Santoro, Instituto Butantan, BRAZIL Received: November 15, 2023; Accepted: February 6, 2024; Published: February 21, 2024 Copyright: © 2024 Mendoza-Roldan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Representative sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank) (accession number OR753376 for gltA; OR755903 to OQ630505 for 16S rRNA; OQ632771 to OQ632773, OR771463 to OR771475 and OR771476-OR771477 for 18S rRNA; OQ672452, OR758867, OR761977, OR761978 and for OR763078 cox1). Funding: D.O. and J.A.M.R. were partially supported by EU funding within the NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors report there are no competing interests to declare.

Introduction Snakes’ (Serpentes: Squamata) perception and interaction with human societies can be contrasting, generating fear and negative feelings (e.g., disgust, repulsion; [1,2]), being merely tolerated or even used for food or economic sources (i.e., snake charming) [3], or considered as new companion animals [4,5]. One of the major threats on snake conservation is the anthropogenic pressure, directly implicated in the decline of snake populations [6,7]. Similarly, habitat and biodiversity loss and climate change represent main threats on snake populations [8–11]. Important threats are also represented by the predation by domestic carnivores (i.e., dogs and cats) and the high density of some wild ungulates, such as wild boars [12–14], as well as the emergence of pathogens (e.g., Ophidiomyces ophiodiicola) within wild populations of snakes [15]. Both factors above are connected, as many pathogens are transmitted by predation, with snakes being intermediate or definitive hosts of parasites, some of which are zoonotic [16]. The relationships and uses by human communities of reptiles is also known as ethnoherpetology [17], which studies the importance of reptiles in different ecological, economic, and cultural contexts [2]. Further investigations were conducted toward integrating One-Health parasitological approaches with ethnoherpetology (i.e., ethnoherpetoparasitology), which allowed to identify the microorganisms and parasites that these animals harbor, as well as the potential risk of zoonotic transmission to snake charmers and vendors in the souks of Marrakech, Morocco [3]. All of the above was assessed in a place where snakes are feared but highly tolerated, given their cultural and economic importance. In Italy, these slithery animals are part of socio-cultural and religious aspects of the country’s history. One of the most ancient and iconic ethnoherpetological rituals across Europe, known as the “festa dei serpari” (also called the ritual of San Domenico), is performed in the small mountainous town of Cocullo, central Apennine (Abruzzo, central Italy) [6,18,19]. This mixed Catholic and pagan ceremony has been performed for centuries during the first days of May, with little to no alterations, consisting of placing four-lined snakes (i.e., E. quatuorlineata) on top of the statue of San Domenico [20]. Soon after, the snake-adorned statue is taken through the small town, in a religious procession with thousands of onlookers in attendance. During the main event, other species of snakes (e.g., western whip snake—Herophis viridiflavus, Aesculapian snake—Zamenis longissimus, juvenile specimens of E. quatuorlineata) are handled by “serpari” (i.e., people that capture and handle the snakes) for thousands of pilgrims and tourists to photograph or interact with them [18,21]. In order to have a good number of snakes for the festival, the “serpari” are formally authorized by relevant authorities to capture snakes alive in the surroundings of the Cocullo municipality from the 19th of March till the 30th of April, after which they are obliged to release them in the same capture sites, within three days following the main event. In Italy, most parasitological studies on snakes have been focused on Cryptosporidium, helminths and ectoparasites in exotic/pet snakes [22–24], with few investigations focused on wild species [25]. In the same context, the introduction of exotic parasites (e.g., Renifer aniarum) in grass snakes (Natrix natrix) and of snake fungal disease (SFC) caused by O. ophiodiicola in dice snakes (Natrix tessellata) stresses the importance of monitoring the health status of wild populations of animals by accurate risk assessment [26–28]. In addition, aside from salmonellosis [29,30], zoonotic parasites of reptiles [16,31], including Reptile Vector-Borne diseases (RVBDs; [32]) have gained interest of the scientific community. Indeed, wild snakes are sentinels for zoonotic agents as they are reservoirs of a plethora of pathogens, playing a role in the life cycle of helminths (i.e., cestodes, nematodes and trematodes), pentastomids and vector-borne pathogens [16]. Ticks such as Ixodes ricinus have been collected from wild four-lined snakes (Elaphe quatuorlineata) from southern Italy, that tested positive for Mediterranean spotted-fever (Rickettsia helvetica), but not for Lyme disease (Borrelia burgdorferi sensu lato [33–35]). Other zoonotic parasites have been identified in free-ranging snakes, such as Spirometra erinaceieuropaei, Mesocestoides and Raillietiella [25,36]. Moreover, efforts have been carried out to assess possible emerging pathogens that could be a threat to the snake populations, such as bacteria [37], and the devastating keratinophilic fungus O. ophiodiicola [27]. Considering all the above, the present study aimed to investigate parasites and microorganisms associated to snakes collected for the “festa dei serpari” ritual, as well as to identify potential zoonotic pathogens that these animals may harbor.

Discussion Ecto- and endoparasites were identified using a morpho-molecular approach from four of the five screened species of snakes collected for the “festa dei serpari” ritual. While most of the identified parasites are specific of reptiles and non-pathogenic (i.e., mites, helminths and protozoa), others transmitted by ticks (e.g., Rickettsia), as well as through fecal-oral transmission (i.e., Cryptosporidium spp., Giardia; Pseudomonas, Proteus vulgaris) have a zoonotic potential. The surveillance performed in this study with the local authorities, allowed to evaluate the parasitic fauna of free-ranging native snakes, which has been until now scarcely investigated or tackled in the Italian ophidic fauna. The species composition of snake population (i.e., E. quatuorlineata, H. viridiflavus, Z. longissimus) is typical of the surroundings of Cocullo municipality, as already observed in previous screenings [27]. On the other hand, the absence of ticks in the snake population from this study may be due to the collection of the snakes during the early spring, where ophidians are less exposed to larvae and nymphs of I. ricinus [67]. Indeed, this tick species is most prevalent in woody areas of central Italy and was previously recorded in four-lined snakes from southern Italy [33]. In addition, immature stages of I. ricinus may prefer other reptile species that are more abundant and fossorial, such as Podarcis or Lacerta lizards [68,69]. The Ixodorhynchidae mites identified herein (H. piger) were described from an unknown snake collected in Florence (central Italy) [44]. Probably, H. viridiflavus is new host for this mite species, as well as Cocullo a new locality. The Ophionyssus species herein identified differs from the common snake mite, O. natricis, which is usually found in captive snakes around the world and may feed also on humans [70]. Moreover, the identified mite specimens morphologically differed from the 16 known species of Ophionyssus [42,43], with low homology in the cox1 nucleotide sequences compared to those of O. natricis deposited in Genbank. Given all the above, further studies are required to elucidate if the Ophionyssus species found is in fact a new species. Nonetheless, H. viridiflavus and E. quatuorlineata represent new hosts for Ophionyssus mites, as well as the Abruzzo region a new locality for Macronyssidae mites of snakes. Although all of the tested mites were negative for pathogens, the molecular detection of Wolbachia sp. in Ophionyssus sp. supports a previous finding in O. natricis from captive Boa constrictor [24], as well as in Ornithonyssus bursa [71]. The finding of Wolbachia in female mites, but not males, may be due to the role this endosymbiont bacterium displays in the reproduction of mites, through male-killing, feminization, and parthenogenesis [72,73]. Furthermore, the diversity and prevalence of the endoparasitic fauna of snakes recorded in this study (39.4%) were higher than that reported for captive snakes from Poland (i.e., 13.7%; [74]) and Italy (i.e., 10.5%; [23]), and lower from another survey conducted also in Italy (56.8%; [75]). Nevertheless, this study represents the first coprological survey of free-raging snakes in the Italian peninsula, without the need of euthanizing or working with recently dead/killed snakes [25,76,77]. Coccidia identified were morphologically similar with Eimeria [78], of mild pathogenicity, commonly observed in wild snakes. In addition, snakes were found infected with helminth eggs similar to Kalicephalus, Rhabdias and Strongyloides [79], as well as oxyurids and Capillaria eggs [80], with the latter being generally found in healthy animals. Overall, the findings of the above parasites may derive from the predation attitude of snakes [81] under different ecological contexts, therefore representing spurious parasites [82], rather than host specific ones. The possibility of having spurious parasites is higher in wild snakes than in those kept in captivity (e.g., 1.4%, 4/283; [82]), given that they actively feed on small preys. Given all the above, future studies are warranted to better distinguish real parasitic fauna of free-ranging snakes from spurious or free-living parasites. Importantly, molecular screening allowed to identify potentially zoonotic parasites and microorganisms, highlighting the need of an integrative approach using morpho-molecular techniques to assess wild animal populations under a One-Health perspective. For the zoonotic protozoa identified with the 5-plex qPCR, confirmatory sequences could only be retrieved for Cryptosporidium, but they were too short for reliable species identification. Zoonotic species, such as Cryptosporidium muris, Cryptosporidium parvum and Cryptosporidium tyzzeri were already identified in captive snakes [83]. Indeed, zoonotic Cryptosporidium species are spurious parasites in snakes, where resistant oocysts may contaminate the environment. Given that the diet of both species of snakes (i.e., E. quatuorlineata, H. viridiflavus) found positive for Cryptosporidium may include also rodents [84,85], further attempts should establish whether the captured snakes of Cocullo harbor zoonotic Cryptosporidium species or Cryptosporidium serpentis. The latter species may cause asymptomatic or chronic infections, being highly pathogenic, infectious, and irresponsive to therapeutic treatment [86]. The 5-plex qPCR may have low specificity and limited discriminatory power for the highly diverse protozoa species in snakes’ feces [62,87]. Moreover, mixed infections with related species of protozoa result in close melting curves hindering the detection of targeted species. Nonetheless, specific investigations on zoonotic pathogens in snakes are warranted considering that G. duodenalis is an important food and waterborne pathogen [88] as well as E. histolytica. The latter has never been reported in snakes [89], which typically host a highly pathogenic reptilian protozoan, Entamoeba invadens, causing necrotic enteritis and hepatitis [90], as well as the less pathogenic species Entamoeba ranarum [90]. Certainly, the integrative approach using morphological and molecular tools further permitted the identification of nematodes belonging to the genera Oswaldocruzia and Rhabdias from cloacal swabs, when fecal samples were not collected [91,92]. These genera of nematodes are potentially pathogenic to snakes and should be actively surveyed to assess their deleterious effects on the free-raging snake populations, also considering the zoonotic potential and emergence of snake-associated human parasites, such as Ophidascaris robertsi neural larva migrans [93]. While it was not possible to perform a definitive identification of zoonotic parasites, the sequences obtained from fecal and cloacal swabs allowed the detection of zoonotic bacteria (i.e., A. xylosoxidans, C. freundii, P. vulgaris, Pseudomonas), which were already detected in snakes, that may act both as reservoirs and spreaders [94,95]. As the bacteria above may be opportunistic pathogens of humans and multi-drug resistant strains have been identified [96], correct biosafety measures should be applied during and after the “festa dei serpari” event. This is mainly due to the fact that, when handled, free-ranging snakes defecate as a defense mechanism [97,98], therefore increasing the risks of contamination with pathogens such as Salmonella [29,99]. Regarding zoonotic vector-borne pathogens, the detection of R. aeschlimannii in Z. longissimus blood suggests exposure to tick bites, further corroborating the potential role of reptiles as reservoirs for Rickettisa spp. [100,101]. Again, molecular positivity for species of the spotted fever group (i.e., Rickettsia monacensis, Rickettsia helvetica) was reported in lizards from Italy [35] and snakes (i.e., Rickettsia asiatica) from Morocco [3]. As R. aeschlimannii and other rickettsiae (i.e., R. monacensis, Rickettsia massiliae) are considered as emerging human pathogens [102], further studies should be conducted to verify the occurrence of this species of Rickettsia, previously detected in Algeria from Hyalomma aegyptium ticks collected from tortoises [103]. The retrieval of the reptile-associated L. tarentolae from snakes in Italy represents new hosts (i.e., E. quatuorlineata, H. viridiflavus) and broadens its geographical distribution northwards, near the Lazio region where it was previously detected in human blood donors and sand flies [104]. Prior surveys from Italy yielded positive molecular results in species of lizards and geckos for L. infantum and L. tarentolae, in urban, peri-urban areas and dog shelters [105]. In addition, L. tarentolae was detected for the first time from cloacal swabs, however, attempts to isolate Leishmania spp. from snakes are warranted given that, thus far L. tarentolae has been isolated only from geckos of the Mediterranean basin [106,107]. To further address the epidemiological picture of Leishmania, entomological surveys are pivotal to describe the species composition of sand flies and address if there is also a sympatric occurrence of both L. tarentolae and L. infantum in the surrounding of the Cocullo municipality, given that L. tarentolae can potentially infect mammals (i.e., humans and dogs) [106]. Given all of the above, the population of snakes around Cocullo may be in part under a “positive” human-snake relationship, where traditional beliefs impact directly on reptile conservation. This, effect has been already studied in other cultural contexts for reptiles such as water monitor lizards in the surroundings of a small village in northern India [108]. As in Cocullo, villagers from the small town of Chak Manik have many beliefs (i.e., protecting the marshlands for their Gods and for the village to thrive), that indirectly have a positive effect on the vulnerable population of reptiles, being mutually beneficial for both the villagers and the reptile species [108]. Although highly subjective, overall health status and condition of the screened snakes was established as “apparently healthy”. Compared to the previous study, where 23 animals had some type of dermal abnormality [27], the three snakes that were herein reported as having skin lesions, were all probably signs of healed trauma or infection. However, given that it was not possible to discard fungal granuloma, annual screening of the collected snake population of Cocullo, assessing fungal, bacterial, and parasitic infections, should be encouraged to have a well-established and consistent surveillance program that will allow for rapid detection of harmful and zoonotic pathogens. Accordingly, results from this study will aid to create strategies to prevent zoonotic transmission of pathogens. Indeed, alongside the established snakes’ population monitoring efforts, pathogens surveillance using a multi-sectorial approach should be also performed annually to assess zoonotic pathogen emergence and their dynamics within the capture population of snakes, that are after released in the environment [109]. Data generated from the present study will be useful for local and national authorities to formulate proper prevention policies specific for the serpari as well as for tourist and pilgrims that participate in the event. For example, education and training of serpari on proper husbandry and protective measures when capturing and handling snakes will aid to reduce the risk of transmission of pathogens to this group of people that are at higher risk, as they are in contact with snakes for over a month [110]. Indeed, educating serpari on proper husbandry and procedures such as quarantine of snakes and the proper use of personal protective equipment (PPE), may reduce the risk of transmission from snake to snake, as well to humans [111]. On the other hand, coordinated policies with local authorities during the event are important to minimize the risk of oral-fecal transmission of pathogens from snakes to tourists. These prevention strategies should be focused on providing hand hygienization/disinfection places, as well as information on why and how to wash their hand after handling a wild animal [112]. Using a One-Health approach to monitor the snake population and reduce the risk of zoonotic transmission will thereby contribute to the conservation of the snakes and the perpetuation of the tradition.

Conclusion Data presented here demonstrate that using an ethnoherpetoparasitological framework to assess the collected ophidian population prior to the annual celebration of the “festa dei serpari” ritual in Cocullo, is a useful approach that allowed for the assessment of the health of the handled snakes, as well as the risks of transmission of zoonotic pathogens present in wild populations. Although snake collected for the ritual harbored reptile-specific and non-pathogenic mites, helminths, and protozoa, the presence of zoonotic pathogens should not be disregarded. This is the case with vector-borne pathogens (e.g., Rickettsia, Leishmania), as well as opportunistic zoonotic pathogens (i.e., Cryptosporidium, Giardia, A. xylosoxidans, C. freundii, P. vulgaris, Pseudomonas) present in the feces of these scaley animals. Thus, snakes collected and showcased in the “festa dei serpari” are optimal sentinels and bioindicators of environmental and ophidian population health, as well as reservoirs of microorganisms that should be controlled through proper biosafety measures when handled by serpari to avoid the risk of zoonotic transmission. Effective public health policies within this unique epidemiological context are advocated, while promoting targeted conservation initiatives, education, and biosafety measures.

Supporting information S1 Video. Festa dei Serpati ritual. Every 1st of may for more than 500 hundred years, the statue of San Domenico is covered with four-lined snakes (Elaphe quatuorlineata), followed by a procession through the small town of Cocullo, Italy. https://doi.org/10.1371/journal.pntd.0011973.s001 (MP4)

Acknowledgments Authors thank Giada Annoscia (UniBa) for her technical work in molecular biology in the laboratories of de Department of Veterinary Medicine of the university of Bari. Authors would like to thank the Serpari, the local community, authorities, and the veterinarian Pasqualino Piro for supporting and rendering possible this study. Authors would like to also thank, Rifcon GmbH for sponsoring since 2016 the professional terraria to house captured reptiles. Authors would like to also acknowledge the Major and the local community of Cocullo for supporting and allowing this annual survey.

[END]
---
[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0011973

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.