Acta Herpetologica 18(1): 45-52, 2023 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-14359 Screening of Ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of San Domenico e dei Serpari (Cocullo, AQ, Italy) Daniele Marini1,2, Ernesto Filippi3,*, Gianpaolo Montinaro4, Francesco C. Origgi5,6 1 Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy 2 Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, 752 36 Uppsala, Sweden 3 via Bagnara Calabra 6, 00178 Rome, Italy 4 Rifcon GmbH, Goldbeckstraße 13, 69493 Hirschberg an der Bergstraße, Germany 5 Institute of Animal Pathology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Läng- gassstrasse 120, 3012, Bern, Switzerland 6 Institute of infectious animal diseases (IIAD), Department of Veterinary Sciences, University of Messina, Viale Giovanni Palatucci sn, 98168 Messina, Italy *Corresponding author. Email: ernesto.flp@gmail.com Submitted on: 2023, 4th February; Revised on: 2023, 4th May; Accepted on: 2023, 8th May Editor: Emilio Sperone Abstract. In the Abruzzi village of Cocullo (Italy), each year, on May 1st, local snake hunters (known as Serpari) dis- play colubrids, captured in the wild, to commemorate the ancient ritual of San Domenico. The ascomycete Ophid- iomyces ophidiicola (Oo) is the causative agent of ophidiomycosis, an emerging disease with sublethal effects. Skin lesions, such as dysecdysis, edematous, crusty or necrotic scales, swellings, nodules, and ulcers, are the most common clinical manifestation of the disease. The pathogen and its associated disease are well characterized in wild snakes in North America, whereas broad screenings of free ranging wild ophidians in Europe are rare. In 2019, as part of a multi-year snake health monitoring project, all the Cocullo ophidians were carefully examined for integumentary affections and those showing signs consistent with ophidiomycosis were dry swabbed on the skin and on any visible cutaneous lesions with a single applicator. The extracted DNA underwent a broad-range panfungal PCR targeting the D1-D2 region, as well as two conventional PCRs specific to the ITS2 and IGS regions of Oo DNA. Twenty-three out of 129 snakes (13/82 Elaphe quatuorlineata; 7/31 Hierophis viridiflavus; 3/15 Zamenis longissumus; 0/1 Natrix helvetica) resulted clinically affected, but no specific Oo genomic DNA was detected by PCR. The Cocullo ritual celebration provided a unique opportunity for the first systematic testing of a large sample size of a local snake community for the monitoring of this pathogen in Italy. Keywords. Ophidiomycosis, Snake Fungal Disease, SFD, snakes, health monitoring, Cocullo, Abruzzi (Italy). Ophidiomyces ophidiicola (Oo) is the etiological agent of ophidiomycosis (also known as Snake Fungal Disease – SFD), a fungal infection of snakes (Lorch et al., 2015). This onygenalean fungus is resistant to various physical and chemical agents (Allender et al., 2015b), and hiber- nacula may represent its environmental reservoir (Camp- bell et al. 2021). Ophidiomyces infection has been asso- ciated with sublethal effects on adults (Agugliaro et al., 2020; Lind et al. 2018a, b; Tetzlaff et al., 2017) and poten- tially lethal outcomes on newborns (e.g., Britton et al., 2019), translating into a potential impact on wild popula- tions’ fitness and a threat to conservation. This emerging 46 Daniele Marini et alii infectious disease occurs with various cutaneous signs as dysecdysis, desquamation, scales abnormalities (e.g., dis- placing), local skin thickening, yellowish/brownish crusts, skin ulcerations, swelling and nodules (revised by Baker et al., 2019), whereas visceral lesions are less frequently recorded. Albeit impacts on different populations seem locally divergent or controversial, Oo has been detected in free-ranging ophidians in most part of North America (Di Nicola et al., 2022). In Europe, samples deriving from wild Coronella austriaca, Hierophis viridiflavus, Natrix helvetica, N. maura, N. natrix, N. tessellata, Vipera berus, V. nikolskii and Zamenis longissimus from UK, Czech Republic, Switzerland, Germany, France, Austria, Hun- gary, Poland, Ukraine, or Italy tested positive with molec- ular methods (Franklinos et al., 2017; Meier et al., 2018; Schüler et al., 2022; Blanvillain et al., 2022; Marini et al., 2023), and a retrospective analysis date back the presence of the fungus in Italy and Switzerland since 1959 (Origgi et al., 2022). In this paper we report the results of an investiga- tion aimed at testing the presence of Oo in a snake com- munity from Central Italy. Data were obtained by snakes captured for a religious ritual (the Catholic cult of San Domenico – of pagan and pre-Christian origins) in the village of Cocullo (Abruzzi). This ceremony takes place in the first days of May, and has remained unchanged for several hundred years. The main feature of this occur- rence is the presence of large numbers of wild-caught snakes by local snake hunters (serpari) during the weeks before the events. This ritual is well known and impor- tant in Abruzzi’s (Italy) culture and history, and it is closely dependent on the local ophidiofauna. In recent years, the ceremony has been accompanied by some sig- nificant conservation actions by the local authorities of Cocullo, due to the increased awareness of the impor- tance of environmental protection, in particular snake conservation. Although no decline of Cocullo’s snakes’ populations has been anecdotally detected in past years, some effects on the reproductive phenology of E. quat- uorlineata and Z. longissimus has been observed (Filippi and Luiselli, 2003) and a few areas surrounding Cocullo are characterized by some disturbance factors for ophidi- ofauna, including a high density of wild boars (regarding this critical issue on snakes see Filippi and Luiselli, 2002). Various species of Colubridae are involved during the cel- ebration, in particular Elaphe quatuorlineata (Pellegrini et al., 2017), one of the largest and more vulnerable spe- cies of snakes in Mediterranean central Italy (Filippi and Luiselli, 2000; Filippi, 2003; Capula and Filippi, 2011), but also Zamenis longissimus and Hierophis viridifla- vus. Snakes are captured by snake hunters from the 19th of March till the 30th of April every year. Since 2010, all snakes captured by serpari are assessed during the 2-3 days preceding the event, that has a fixed date on May 1st (overall n = 1300 snakes were registered and checked from 2010 to 2023, Filippi and Montinaro, in prep.): a scientific committee (composed by EF and GM in col- laboration with a veterinarian) records the captured spe- cies, biometric data (weight, snout-vent length [SVL]), sex, age class (juvenile, subadult, adult), site of capture of the ophidians brought by snake hunters. Moreover, PIT tags are checked or implanted, and a physical examina- tion is carried out in addition to a swab for bacteriologi- cal analyses (processed by IZS - Istituto Zooprofilattico Sperimentale Abruzzo e Molise from 2015 – e.g., Filippi et al. 2010). Then, at the end of the rite, or in any case within seven days, snakes are released by Serpari in the same place where they were captured. In 2019, on 29th and 30th of April, we also conducted a focused survey to assess the presence of Oo (in 2020 and 2021 the rite did not take place due to the Covid-19 pandemic). All snakes underwent an additional physi- cal examination. During this investigation, a particular attention was given to macroscopic clinical signs consist- ent with ophidiomycosis (Fig. 1). Based on this, a binary value according to Hileman and colleagues (2018) was assigned to each snake: “0” (absence of signs consist- ent with ophidiomycosis); “1” (presence of signs consist- ent with ophidiomycosis). The clinical signs from the snakes categorised as “1” (clinically affected) were care- fully documented, and the grade of infection severity was retrospectively calculated following the Infection Sever- ity Score (ISS) proposed by Baker and colleagues (2019) (Table 1). A single sterile cotton-tipped applicator with wooden stick (Aptaca Spa, Canelli, Italy) was used to (dry) swab each individual belonging to the apparently affected group, and then placed in 20 ml plastic tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany). The snakes were swabbed with moderate pressure 10 times along the entire dorsal surface, ventral surface, head, and, additionally, ≥ 10 times on each suspected lesion (see Di Nicola et al. 2022; Marini et al., 2023). In cases in which scales and/or lesions naturally exfoliated during swabbing (i.e., abnormal scales partially detached or scales adjacent to skin lesions) or pieces of exuviae detached (due to dys- ecdysis), these tissues were stored together with the swab from the same individual in the same 20 ml vial. The tubes were stored at +4 °C until shipment. Each snake was handled by the veterinarian carrying out the swab (DM) with new disposable nitrile or latex gloves and all the equipment eventually used (hooks, forceps, scale) was disinfected with 95% denatured ethanol or 5% sodi- um hypochlorite solution. The DNA has been extracted by placing each swab (with or without tissue) in a 1,5 47Ophidiomyces screening in Cocullo snake community ml safe-seal tube with one aliquot (500 µl) of lysis buff- er (0.1 M Tris–HCl, pH 8.5, 0.05% Tween 20, 0.24 mg/ ml proteinase K), then placed in a heat block first at 60 °C for 1 hour and after 95 °C for 15 minutes. Afterward, 10 µl of each lysate were diluted in 90 µl of nuclease free water. A broad-range standard panfungal PCR (amplify- ing the D1-D2 region of the large subunit [LSU – 28S] of the ribosomal RNA [rRNA] gene complex – Borman et al., 2006) was performed following Frankinos et al. (2017). Moreover, two different set of primers described by Bohuski and colleagues (2015), targeting specific regions of Oo genome – ITS2 (internal transcribed spacer region 2) and IGS (intergenic spacer region) within the rRNA gene – were employed for conventional PCR assays (Origgi et al., 2022). Each conventional (qualitative) PCR assay has been run in 30 μl amplification mixture com- posed of 3 μl of PCR Buffer, 0.6 μl dNTP mix (10 mM each), 0.75 μl of each primer (100 mM), 0.3 μl of Taq Polymerase, 2.5 μl of DNA template (diluted lysates), 3.8 μl of MgCl2 (25 mM), and 18.3 μl of water (Solys Bio- dyne, Luzerna Chem, Lucerne, CH). The reactions were carried out as follows: initial denaturation (95 °C for 3 min) followed by 35 cycles including 30 s at 95 °C (dena- turation), 30 s at 52 °C for ITS and 50°C for IGS (anneal- ing), 30 s at 72 °C (elongation). A final extension at 72 °C for 10 min followed. Lastly, 5µl of the PCR product were resolved on a 2% agarose gel by electrophoresis and visu- alized under UV light. A total of 129 snakes (adults, subadults and juve- niles) were brought by serpari and examined by the sci- entific committee. Ophidians belonged to the following species: Elaphe quatuorlineata (n = 82), Hierophis vir- idiflavus (n = 31); Zamenis longissimus (n = 15), Natrix helvetica (n = 1). Twenty-three snakes out of 129 (17.8%) Fig. 1. Representative lesions of individuals grouped in category “1” (presence of signs consistent with ophidiomycosis): (A) EQ21, Elaphe quatuorlineata showing erosions of rostral, right internasal, nasal, preocular, ocular and supraocular scales and displaced dorsal scales; (B) EQ46, Elaphe quatuorlineata exhibiting multifocal swollen, eroded and hyperaemic (dorsal and ventral) scales associated with crusts and dysecdysis; (C) HV28, Hierophis viridiflavus with hyperaemia, erythematous skin, and retained exuvium in the gular region; (D) HV04, Hierophis viridiflavus showing wrinkled, depressed and crusty dorsal scales. 48 Daniele Marini et alii Ta bl e 1. I nd iv id ua ls c at eg or is ed a s cl in ic al ly a ffe ct ed ( ca te go ry “ 1” ) an d sa m pl ed a t C oc ul lo ’s fe st iv al i n 20 19 . F or e ac h sn ak e th e ta bl e sh ow s th e id en tifi ca tio n co de , s pe ci es , s ex a nd ag e cl as s, s no ut -v en t le ng th ( SV L) , w ei gh t, ty pe o f co lle ct ed s am pl e, le si on s co re s (t yp e, lo ca tio n, n um be r, co ve ra ge ) an d th e re la tiv e In fe ct io n Se ve ri ty S co re ( IS S) , a nd t he d es cr ip tio n of g ro ss s ig ns . M : a du lt m al e. F : a du lt fe m al e. S A F: s ub ad ul t f em al e. S : d ry s w ab . T : t is su e. B C S: b od y co nd iti on s co re . Id Sp ec ie s Se x an d ag e SV L (c m ) W ei gh t (g ) Sa m pl e ty pe Le si on ty pe sc or e Le si on lo ca tio n sc or e Le si on nu m be r sc or e Le si on co ve ra ge sc or e IS S (I nf ec tio n Se ve ri ty Sc or e) G ro ss s ig ns d es cr ip tio n EQ 21 El ap he qu at uo rl in ea ta SA F 10 7 30 6 S 3 3 3 3 12 C ru st y er os io ns o f r os tr al , r ig ht in te rn as al , n as al , p re oc ul ar , o cu la r an d su pr ao cu la r sc al es . C ru st y, d ry , d us ty , d is lo ca te d an d (s om et im es ) er od ed sc al es m ul tif oc al ly a lo ng th e do rs um . B ro w ni sh a nd d us ty c ru st o f t he ta il (> 3x 2c m ). H V 04 H ie ro ph is vi ri di fla vu s M 89 26 4 S 2 1 3 3 9 D es qu am at io ns w ith d us ty a sp ec t. C on ca ve , w ri nk le d or c ru st y do rs al s ca le s. Tr au m at ic a nd c ru st y le si on s of v en tr al s ca le s. EQ 24 El ap he qu at uo rl in ea ta M 13 9 10 86 S 2 2 3 2 9 Ec to pi c or c ru st y or d is lo ca te d do rs al a nd ta il sc al es . R et ai ne d sh ed b et w ee n su pr ao cu la r, oc ul ar a nd p os to cu la r sc al es . F oc al d is co lo ra tio ns . L ow B C S. H V 11 H ie ro ph is vi ri di fla vu s M 95 26 0 S 2 2 2 2 8 R et ai ne d sh ed in th e pa ri et al a nd d or sa l s ca le s of h ea d re gi on . C ru st y, d us ty an d/ or r ai se d do rs al s ca le s. L ig ht d eh yd ra tio n. H V 12 H ie ro ph is vi ri di fla vu s M 87 29 6 S 2 2 2 2 8 C oa le sc en t d es qu am at io ns o f h ea d an d tr un k sc al es . C on ca ve o r cr us ty d or sa l sc al es . EQ 27 El ap he qu at uo rl in ea ta F 14 2 85 4 S 2 2 2 3 9 Bi lo be d lo os e sw el lin g (> 4 x1 ,5 cm - p re su m pt iv el y su bc ut an eo us n od ul e) on th e le ft la te ra l r eg io n of th e tr un k. M od er at e nu m be r of e ct op ic a nd di sl oc at ed d or sa l s ca le s. W ri nk le d sc ar b et w ee n ro st ra l a nd in te rn as al s sc al es . Z L0 1 Z am en is lo ng is si m us M 99 19 4 S 2 3 1 2 8 Tu m ef ac tio n on th e le ft si de o f t he u pp er ja w . A t b uc ca l i ns pe ct io n, th e m uc os a w as fo un d hy pe ra em ic , h ae m or rh ag ic a nd s w el le d. Z L0 7 Z am en is lo ng is si m us M 10 5 39 6 S 2 2 2 2 8 D es qu am at io n w ith a s lig ht ly c ru st y an d w ri nk le d as pe ct o n th e ri gh t l or ea l, pr eo cu la r an d su pr al ab ia l s ca le s. D is lo ca te d, c on ca ve a nd e ct op ic d or sa l sc al es . H V 15 H ie ro ph is vi ri di fla vu s M 96 32 0 S 1 2 2 2 7 A br as io n be tw ee n ro st ra l a nd in te rn as al s sc al es . D is lo ca te d, e ct op ic , w ri nk le d or a bs en t d or sa l s ca le s. EQ 39 El ap he qu at uo rl in ea ta F 15 0 10 00 S 2 2 2 1 7 C ru st y le si on o n ri gh t s op ra oc ul ar s ca le . D is lo ca te d do rs al s ca le . EQ 40 El ap he qu at uo rl in ea ta M 12 4 68 2 S 1 1 2 2 6 Fo ca l d is co lo ra tio ns . C on ca ve d or sa l s ca le s. EQ 46 El ap he qu at uo rl in ea ta F 11 2 38 0 S, T 3 1 3 3 10 D ys ec dy si s an d re ta in ed m ou lts in m an y lo ca tio ns . T um ef ac te d, e ro de d an d hy pe ra em ic ( do rs al a nd v en tr al ) sc al es . S ev er al y el lo w is h- br ow ni sh c ru st s le si on s in th e do rs al a nd v en tr al r eg io n of th e tr un k, m ul tif oc al p at te rn . So m e sw ol le n sc al es o r cr us ts u nd er ly in g no du la r fo rm at io ns . L ow B C S an d m us cu la r w ea kn es s. EQ 50 El ap he qu at uo rl in ea ta M 11 6 44 4 S 1 2 2 2 7 M ul tif oc al d is co lo ra tio ns . W ri nk le d do rs al s ca le s. I rr eg ul ar c au da l e dg es o f ve nt ra l s ca le s. EQ 54 El ap he qu at uo rl in ea ta F 13 8 96 6 S 2 2 2 3 9 C ru st y le si on s on d or sa l s ca le s. C on ca ve a nd d is lo ca te d do rs al s ca le s. Er yt he m at ou s an d hy pe ra em ic v en tr al s ca le s. N od ul ar fo rm at io n on th e ta il (> 1 x1 cm ). 49Ophidiomyces screening in Cocullo snake community Id Sp ec ie s Se x an d ag e SV L (c m ) W ei gh t (g ) Sa m pl e ty pe Le si on ty pe sc or e Le si on lo ca tio n sc or e Le si on nu m be r sc or e Le si on co ve ra ge sc or e IS S (I nf ec tio n Se ve ri ty Sc or e) G ro ss s ig ns d es cr ip tio n EQ 55 El ap he qu at uo rl in ea ta M 11 7 55 6 S 2 1 3 3 9 Sc at te re d da rk s ca rs . D is lo ca te d, c ru st y, w ri nk le d or a bs en t d or sa l s ca le s. H V 21 H ie ro ph is vi ri di fla vu s M 96 34 8 S 2 1 3 3 9 Br ow ni sh -y el lo w is h m oi st fr es h cr us ts d or sa lly a nd v en tr al ly o n th e ta il. C on ca ve , c ru st y or a bs en t d or sa l s ca le s. H V 23 H ie ro ph is vi ri di fla vu s M 93 28 8 S 3 1 2 2 8 D is lo ca te d an d co nc av e do rs al s ca le s. P at ch es o f d is co lo ra tio ns . T um ef ac tio n (> 1 x1 c m ) on th e le ft si de o f t ai l ( ad ja ce nt e ry th em at ou s an d er os iv e ve nt ra l sc al es ). H V 28 H ie ro ph is vi ri di fla vu s M 10 3 34 2 S, T 2 2 2 3 9 D ys ec dy si s. R et ai ne d ex uv iu m a t l ev el o f s no ut a nd c hi n. H yp er ae m ic a nd er yt he m at ou s sk in in th e gu la r re gi on . D es qu am at io n an d w ri nk lin g of d or sa l sc al es . T w o no du la r fo rm at io ns ( 0. 5x 0. 5 cm ) on th e tr un k. I rr eg ul ar c au da l ed ge s of v en tr al s ca le s. Z L1 3 Z am en is lo ng is si m us F 79 16 2 S, T 3 1 3 2 9 C ru st y, r ai se d or c on ca ve d or sa l s ca le s. R et ai ne d sh ed . F oc al e ro si ve fr es h le si on o n ve nt ra l s ca le s. M ul tif oc al c ru st s on c au da l t ru nk a nd ta il. EQ 58 El ap he qu at uo rl in ea ta M 14 0 10 40 S 2 3 3 2 10 Li gh t m ul tif oc al d is co lo ra tio n. C ru st y, d ry o r di sl oc at ed d or sa l s ca le s. Th re e cr us ty le si on s on th e ve nt ra l s ca le s (o ne o n th e cl oa ca ). EQ 62 El ap he qu at uo rl in ea ta M 13 9 10 88 S 3 1 2 3 9 D ry o r di sl oc at es d or sa l s ca le s. S w el lin g an d hy pe ra em ia o r er os io n an d of ve nt ra l s ca le s cl os e to c lo ac a. O ne n od ul ar fo rm at io n at tr un k le ve l ( 1x 1 cm ), an d on e at ta il le ve l ( 0. 5x 0. 5 cm ). EQ 64 El ap he qu at uo rl in ea ta M 13 4 79 0 S 2 1 2 2 7 D is lo ca te d an d ec to pi c do rs al s ca le s. D es qu am at io ns . C ru st y le si on o f t he ta il. EQ 69 El ap he qu at uo rl in ea ta M 13 6 85 8 S 2 1 2 2 7 D iff us ed d ar k di sl oc at ed a nd w ri nk le d do rs al s ca le s. Th re e no du la r fo rm at io ns o n th e tr un k (< 1 x1 cm ). D oc ke d ta il. 50 Daniele Marini et alii showed signs consistent with a fungal dermatitis and were assigned to the category “1” (apparently affected): Elaphe quatuorlineata (n = 13; 15.8%), Hierophis viridi- flavus (n = 7; 22.6%); Zamenis longissimus (n = 3; 20%). Table 1 reports all the clinically affected ophidians and the macroscopic signs that allow ranking these individu- als in category “1”, as well as each category calculated for counting the individual ISS. Among all the individu- als, the ISS varied between 6 and 12, being 9 the median score. No influence of the species on the ISS was found (χ2 = 11.49, P = 0.32, df = 10). Cluster analysis of the ISSs – with and without normalization of the lesion coverage on the individual weight – did not reveal any particular trend or clustering (data not shown). A total of 23 swabs (and 3 tissues linked to one of them – Table 1) have been collected. After DNA extraction, for every sample (with or without tissue) a conventional PCR was carried out for each of the three targeted region of Oo (D1-D2, ITS2 and IGS regions – 23 samples x 3 reactions = 69 results). No product of consistent size was observed on agarose gel from each PCR electrophoresis (0/69). The number of category “1” individuals versus the number of catego- ry “0” individuals of each species did not differ statisti- cally (χ2 = 0.89, P = 0.64, df = 2). The presence of clinical signs in adult E. quatuorlineata appeared to be associated with weight (ncat1 = 12, x = 812.00 + 247.01; ncat0 = 63, x = 689.02 + 174.49; t-test = 2.09 P = 0.04) but not with sex (Fisher test P = 0.68) and SVL (ncat1= 12, x = 131.42 + 10.67; ncat0 =64, x = 127.88; t-test = 0.98, P = 0.33). SVL and weight were correlated in both apparently clini- cally healthy (r61 =0.74, P < 0.01) and clinically affected individuals (r10 =0.92, P < 0.01). No females with clinical signs were observed in H. viridiflavus and, among males, the number of category “1” adults did not appear to be related to SVL (ncat1= 7, x = 94.14 + 5.24; ncat0 = 13, x = 91.65 + 6.14; t-test = 0.91, P = 0.38). SVL and weight did not positively correlate in clinically affected (r5 =0.62, P > 0.05) while these morphometric parameters were cor- related in unaffected individuals (r11 = 0.82, P < 0.05). Three adults of Z. longissimus (1 female and 2 males) out of 15 were grouped in category “1”. Oo genomic DNA was not detected in any of our samples (n= 23, observed prevalence 0%, Bayesian 95% credible intervals: 0.00- 0.14). Considering the increasing number of ophidiomyco- sis reports in Europe, a standardised monitoring for snake communities is warranted. To the best of our knowl- edge, this is the first systematic testing of a large sample size of a local snake community for the monitoring of O. ophidiicola in Italy. We investigated only individu- als with signs consistent with a fungal dermatitis because the swabs coming from these ophidians are more likely to result (true) positive compared to those showing no lesions (Hileman et al. 2018; Long et al. 2019). Accord- ing to our data, none of the species studied shows an obvious high incidence of clinical signs compared to the other species and, within the same species, no particular trends emerged between clinically affected individuals versus clinically not affected ones and parameters as sex, SVL and weight. The used ISS was a helpful tool to char- acterize the severity of the infection of each individual, independently of the limitations associated with the lack of positive samples in our study. According to our expe- rience, a normalization of the lesion coverage to the size of the animal is recommended (e.g., weight, surface – see Blanvillain et al., 2022). No evidence of the presence of Oo DNA was revealed by PCR. However, PCR negativity is consistent with either the actual absence of the target DNA sequence or its presence under the limit of detec- tion. Furthermore, eventual inhibitors could also ham- per the PCR results. Accordingly, we cannot rule out the occurrence of some false negative. Lastly, the snakes were sampled once, and repeated sampling of the same indi- viduals was shown to significantly reduce the probability of a false-negative (Hileman et al. 2018). Hence, in order to detect eventual false negative and increase sensitivity such screening should be improved by performing multi- ple re-samplings (e.g., 3-5 swab applicators - Hileman et al. 2018; Marini et al., 2023). Also, the use of real-time (quantitative) PCRs instead of conventional (qualita- tive) ones would improve the detection of false negatives from swab samples (Allender et al., 2015a). This fungus might occur in all the temperate regions around the globe (Burbrink et al., 2017) and snake susceptibility may vary according to phylogenetic and ecological factors (Haynes et al., 2020). The actual natural history of the colonization of this fungus is still unclear. The possible introduction of the fungus into North America by pathogen pollution has been suggested (Ladner et al., 2022) along with evidenced of the presence of both the American (Switzerland) and the European clade (Italy) in the European continent for more than 60 years (Origgi et al., 2022). Therefore, it is essential to shed lights on the distribution of this fungus in European continent along with its associated (clade- specific). Accordingly, it is important to carry out screen- ing in Italian territories, implementing what was started in Cocullo. On the other hand, the monitoring of the possi- ble presence of Oo extends and enriches the health moni- toring and conservation actions in place at Cocullo since 2010. In particular, to reduce the potential risk of disease and to ensure an excellent standard of handling and keep- ing of wild ophidians, a vademecum on snakes’ manage- ment in terraria has been published and delivered to the Serpari. Additionally, professional terraria have been allo- 51Ophidiomyces screening in Cocullo snake community cated to properly house the snakes every year, and dedi- cated exhibit with numerous environmental education and training activities concerning snakes are carried out for the thousands of tourists attending the ritual every year. The annual monitoring of the ophidians involved in the Cocullo ritual will provide a great opportunity for collecting baseline data critical to assess the population health of the local snake community, which goes beyond the specific Oo screening, and which represents a para- digmatic example of how cultural traditions, citizen sci- ence and conservation may come all together. ACKNOWLEDGEMENTS Handling and capture by Serpari, as well as by scien- tific committee, is allowed under National authorizations [National law DPR 357/97; permit was granted by Italian Ministry of Environment (MATTM n. prot. 6265/2017 PNM and n. prot. 24601/2020 PNM). We thank the vet- erinarians Gian Lorenzo D’Alterio (from 2010 to 2014) and Pasqualino Piro (from 2014 to date), IZS Lazio e Toscana (I.T. Sez. Viterbo from 2010 to 2013), IZS Abruzzo e Molise for cooperation in conducting clinical examinations and bacteriological analyses of snake com- munity from Rito di San Domenico. We are grateful to Rifcon GmbH that from 2016 sponsored every year pro- fessional terraria to house captured reptiles. We would like to acknowledge the Major, the municipality and the people of Cocullo to let us keep going these conservation actions and to keep alive this ancient ritual in the respect of nature. We are grateful to Ursula Sattler (University of Bern) for her advice on laboratory diagnostics and to Matteo Oliveri for his suggestions on the initial research concept. REFERENCES Agugliaro, J., Lind, C.M., Lorch, J.M., Farrell, T.M. (2020): An emerging fungal pathogen is associated with increased resting metabolic rate and total evapo- rative water loss rate in a winter‐ active snake. Funct. Ecol. 34: 486-496. Allender, M.C., Bunick, D., Dzhaman, E., Burrus, L., Maddox, C. (2015a): Development and use of a real- time polymerase chain reaction assay for the detec- tion of Ophidiomyces ophiodiicola in snakes.  J. Vet. Diagn. 27: 217-220. 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