Introduction
Cylicospirura Vevers 1922 (Spiruroidea: Spirocercidae Chitwood and Wehr 1932) is a genus of parasitic nematodes of wild and domesticated felids, including domestic cats, bobcats, cougars, wildcats, or ocelots (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024), although other animal orders have been demonstrated to be infected with this genus (Ladds et al. Reference Ladds, Sammons and Beveridge2006). Cylicospirura spp. are widely distributed since they have been reported in different geographical locations from the Americas, Africa, Oceania, and Europe (Diakou et al. Reference Diakou, Migli, Dimzas, Morelli, Di Cesare, Youlatos, Lymberakis and Traversa2021; Junker et al. Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013; Ladds et al. Reference Ladds, Sammons and Beveridge2006; Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023). Moreover, these parasites produce distinctive nodular lesions in the animal’s gastric mucosa (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020). In here, adult male and female nematodes develop, copulate, and females later shed their eggs through a fistulated opening of the nodule to the gastrointestinal tract (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024). Based on their biological and ecological similarities to members of the Spirocercidae family, such as Spirocerca lupi (Rudolphi 1809) (Rojas et al. Reference Rojas, Dvir and Baneth2020), it has been proposed that Cylicospirura eggs are ingested by coprophagous beetles where larval development occurs. Then, definitive hosts might become infected after predation of paratenic hosts. This and other aspects of the worm’s pathology, epidemiology, and ecology are poorly understood (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011). However, it is generally accepted that the Cylicospirura genus is highly diverse, with 11 described species restricted to felid hosts.
Eleven Cylicospirura spp. have been described in the last century, including Cylicospirura subaequalis (Molin 1860), Cylicospirura felineus (Chandler 1925) (Pence et al. Reference Pence, Samoil and Stone1978), Cylicospirura heydoni (Baylis 1927), Cylicospirura petrowi (Sadykhov Reference Sadykhov1957), Cylicospirura skrjabini (Kozlov, Owsjukova, and Radkewitch 1964), Cylicospirura crocutae (Junker and Metafchiev Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013) (Junker et al. Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013), and Cylicospirura phili (Junker and Mutafchiev Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024) (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024). Of these, only C. subaequalis and C. felineus have been reported in the Americas, namely in Brazil and the USA. These two species can be differentiated both morphologically and molecularly. At the molecular level, few sequences of this genus have been deposited in genetic databases; however, C. felineus and C. subaequalis can be differentiated with cytochrome oxidase subunit 1 (cox1) fragments which allow the separation from C. petrowi (Rojas et al. Reference Rojas, Dvir, Farkas, Sarma, Borthakur, Jabbar, Markovics, Otranto and Baneth2018).
Cylicospirura spp. have a host specificity towards felids. For instance, C. subaequalis (Vieira et al. Reference Vieira, Muniz-Pereira, Souza-Lima, Marun Rocha and Luque2017) and C. felineus (Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023) were reported in jaguaroundis, Herpailurus yagouaroundi, from Brazil based on histopathological, morphological, and ecological evidence. In addition, C. felineus was detected in Geoffroy’s cat, Leopardus geoffroyi (Gallas et al. Reference Gallas, da Silveira and Périco2014), and cougars, Puma concolor, also in Brazil (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020), as well as in feral domestic shorthair cat, Felis catus (Crossland et al. Reference Crossland, Hanks, Ferguson, Kent, Sanders and Del Piero2015), and bobcats Lynx rufus in the USA (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011). Moreover, cougars from Brazil and the USA have been found with C. subaequalis at a high prevalence of 20% (1/5) (Benatti et al. Reference Benatti, De Santi, Werther, Tebaldi and Hoppe2021) and 73% (117/160) (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011). The last study performed extensive histopathological observations of the animal’s lesions, as well as species confirmation with cox1 analysis (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011).
Previous reports on Cylicospirura have employed various strategies to identify specimens at the genus or species level, each with important epidemiological implications. Some studies have relied solely on coprological methods to detect eggs in faecal samples (Diakou et al. Reference Diakou, Migli, Dimzas, Morelli, Di Cesare, Youlatos, Lymberakis and Traversa2021; Mukarati et al. Reference Mukarati, Vassilev, Tagwireyi and Tavengwa2013; Napoli et al. Reference Napoli, Anile, Arrabito, Scornavacca, Mazzamuto, Gaglio, Otranto, Giannetto and Brianti2016), while others have combined macroscopic examination of worms with histopathological analysis of associated lesions (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020). In contrast, molecular tools have been used in certain cases to achieve species-level identification (Bisterfeld et al. Reference Bisterfeld, Raulf, Waindok, Springer, Lang, Lierz, Siebert and Strube2024; Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011). In this sense, integrative taxonomy combines histopathological, morphometric, molecular, and ecological data to reduce the risk of taxonomic misclassification and synonymisation. Consequently, this approach has emerged as a powerful tool for species description and delimitation, contributing to a steady increase in the number of newly described helminth species over the past 25 years (Rojas et al. Reference Rojas, Bass, Campos-Camacho, Dittel-Meza, Fonseca, Huang-Qiu, Olivares, Romero-Vega, Villegas-Rojas and Solano-Barquero2025).
Since Cylicospirura spp. can affect both domestic and wild felids, studying the genus’s diversity, distribution, and the pathology it induces in its hosts, is highly relevant. In this context, C. felineus infections have been reported in domestic cats from South Africa (Junker et al. Reference Junker, Vorster and Boomker2006), Italy (Ibba et al. Reference Ibba, Lepri, Veronesi, Di Cesare and Paltrinieri2014), and the USA (Crossland et al. Reference Crossland, Hanks, Ferguson, Kent, Sanders and Del Piero2015), highlighting the potential spillover from wild animals to domestic species. This may result from adaptation to phylogenetically related hosts, as observed in other parasites of wild and domestic animals (Matamoros-Suarez et al. Reference Matamoros-Suarez, Montenegro, Villalobos-Brenes, Llaberia-Robledillo, Solano-Barquero, Baneth, Balbuena and Rojas2025), as well as from anthropogenic factors such as changes in land use, habitat fragmentation, and urbanization, which facilitate parasite spillover between wild and domestic populations (Baker et al. Reference Baker, Mahmud, Miller, Rajeev, Rasambainarivo, Rice, Takahashi, Tatem, Wagner, Wang, Wesolowski and Metcalf2022; Weinstein and Lafferty Reference Weinstein and Lafferty2015).
Materials and methods
Collection of samples and pathological analysis of lesions
Three yagouaroundis (Herpailurus yagouaroundi) (Fig. 1A and 1B) were transferred to a rescue centre due to clinical disease in 2019, 2022, and 2023 under resolution SINAC-ACC-OA-re1389-2022. Upon examination, euthanasia was elected. Two of these animals were polytraumatised most likely by a car hit and the other was diagnosed with fungal meningitis. Gross pathological examination of all animals consistently revealed intramural gastric nodules, ranging from 1 to 3 cm in diameter, protruding into the gastric lumen from the submucosa and characterised by a mucosal pore (Fig. 2A). The nodules extended into the mucosa and contained multiple, red-coloured nematodes (Fig. 2C). Parasites were preserved in 70% ethanol for subsequent morphological and molecular analyses. At least one complete nodule per animal was fixed in 10% buffered formalin and processed for histopathological analysis by standard haematoxylin and eosin staining (Zachary and McGain Reference Zachary and McGain2012).
Morphometric assessment of specimens
Ten females and nine males were cleared with lactophenol for 8 hours. After this, specimens were mounted in Canada balsam for the observation of the following structures in females: total body length and width; stoma diameter; buccal capsule length and width; distance of oesophageal valve to the anterior end; oesophageal valve diameter; buccal capsule teeth length; teeth slit length; oesophagus total length; muscular and glandular oesophagus length and ratio; percentage of oesophagus length to total body length; and distance of the vulva to the anterior end. In males, all these parameters, except for the latter, were measured. In addition, the size of the major and minor spicule, distance of cloaca to the posterior end, the distance of the posterior end of the gubernaculum to the posterior of the worm’s body, and the number and disposition of cloacal papillae were recorded. These characteristics were pictured and measured using the AxioCam coupled to an Olympus light microscope. Furthermore, eggs collected from the female’s uterus after spontaneous release were also measured. Arithmetic mean and standard deviations were calculated for each morphological character and compared to other Cylicospirura spp. (Table 1). Line drawings were made with the XP-PEN Deco 03 drawing tablet in InkScape v1.2. Two versions of the drawings were provided: one fully coloured and another in grayscale, available in Supplementary Figure S1.
Comparison of female and male measurements of Cylicospirura wishkai sp. nov. with other Cylicospirura spp
Species similarity was evaluated using subsets of morphometric characters, some of which were sex specific. Given that several characters were inconsistently reported between sexes in the reference literature (Table 1), analyses were performed separately by sex. In those character measurements reported as ranges, the arithmetic mean of the lower and upper limits was taken as a representative value, with the limitation that these values may not be uniformly distributed. Compared species included Cylicospirura wishkai sp. nov. (CW) and seven congeners: C. crocutae (CCr) (Junker et al. Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013); C. felineus derived from two independent reports: sensu Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020 (CF_Alm) found in a Puma concolor from Brazil (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020), and sensu Pence et al. Reference Pence, Samoil and Stone1978 found in a Lynx canadensis from Canada, 1978 (Pence et al. Reference Pence, Samoil and Stone1978); C. felinea (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024); C. pardalis (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024); C. petrowi (CPe) (Sadykhov Reference Sadykhov1957); C. phiri (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024); and C. subaequalis (Waid and Pence Reference Waid and Pence1988). C. advena was not included as this species lacked information on most of the characters.
Morphological characters with more than two unreported values across taxa were removed from the analysis. In addition, characters with exactly one missing value in only one species were imputed with the arithmetic mean value. All measures were standardised to z-scores. Pairwise Euclidean distances computed on these standardised matrices were summarised by agglomerative hierarchical clustering with Ward’s minimum variance criterion. The resulting dendrograms represented overall morphometric similarity between taxa. To visualize the contribution of individual traits, standardised values were also plotted into radar or spider diagrams including all species on a single polar coordinate grid. All analyses were performed in Python 3.11 using the pandas, NumPy, SciPy, and Matplotlib packages for data clustering and visualization.
Scanning electron microscopy of specimens
The anterior and posterior ends of four worms were cut with a sterile blade and processed as previously indicated (Rojas et al. Reference Rojas, Bass, Campos-Camacho, Dittel-Meza, Fonseca, Huang-Qiu, Olivares, Romero-Vega, Villegas-Rojas and Solano-Barquero2025). Briefly, worms were initially fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffer 0.1 M, pH 7.4 for 48 h. Then, specimens were washed with phosphate buffer (0.1 M, pH 7.2), post-fixed with 1% osmium tetroxide (OsO4), washed with distilled water, and dehydrated using ethanol. Fixed specimens were dried using a critical point dryer model EM CPD300 (Leica, Wetzlar, Germany) and mounted on aluminium holders with a carbon double-sided adhesive tape. Samples were sputter-coated with a gold/palladium target using an EMS 150R ES sputter coater (Electron Microscopy Sciences, Philadelphia, USA) and observed in a Scanning Electron Microscope JSM-6010LA (Jeol Ltd, Tokyo, Japan) at 15kV accelerating voltage (Rojas et al., Reference Rojas, Bass, Campos-Camacho, Dittel-Meza, Fonseca, Huang-Qiu, Olivares, Romero-Vega, Villegas-Rojas and Solano-Barquero2025).
Molecular analysis of specimens
DNA was extracted from ten specimens with the DNeasy Blood & Tissue kit (Qiagen®, Hilden, Germany) according to the instructions of the manufacturer. Quality and concentration of the obtained DNA were verified in a NanoDrop® (ThermoFisher Scientific, Waltham, Massachusetts, USA). Then, PCRs were run to amplify fragments of the cytochrome oxidase subunit 1 (cox1), internal transcribed spacer 1 (ITS1), and 18S ribosomal DNA (18S rDNA). Accordingly, NTF (5′-TGATTGGTGGTTTTGGTAA-3′) and NTR (5′-ATAAGTACGAGTATCAATATC-3′) primers (Casiraghi et al. Reference Casiraghi, Anderson, Bandi, Bazzocchi and Genchi2001) were used to amplify a 5′ fragment of the cox1 of nematodes and JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB45 (5′-TAAAGAAAGAACATAATGAAAATG-3′) (Bowles et al. Reference Bowles, Blair and McManus1992) for a contiguous fragment, generating an 800 bp fragment. In addition, 650 bp ITS1 fragments were obtained after amplification with rDNA2 (5′-TTGATTACGTCCCTGCCCTTT-3′) (Vrain et al. Reference Vrain, Wakarchuk, Lévesque and Hamilton1992) and rDNA1.58S (5′-ACGAGCCGAGTGATCCACCG-3′) (Cherry et al. Reference Cherry, Szalanski, Todd and Powers1997) primers and 18S rDNA ~650 bp fragment with 18S-F (5′-CGCGAATRGCTCATTACAACAGC-3′) and 18S-R (5′-GGGCGGTATCTGATCGCC-3′) primers (Floyd et al. Reference Floyd, Rogers, Lambshead and Smith2005). All reactions were run under conditions explained before (Rojas et al. Reference Rojas, Sanchis-Monsonis, Alic, Hodzic, Otranto, Yasur-Landau, Martinez-Carrasco and Baneth2018). Products were run in 1.5% SYBR Safe-stained agarose gels and imaged with a ChemiDoc MP Imaging System (BioRad®, Hercules, California, USA). Amplicons were Sanger sequenced at Macrogen Inc., South Korea.
Obtained sequences were visually inspected for low-resolved nucleotides and aligned with other Cylicospirura spp. available in GenBank®, i.e. C. petrowi, C. felineus, or C. subaequalis using MEGA7 (Kumar et al. Reference Kumar, Stecher and Tamura2016) and the MUSCLE algorithm (Edgar Reference Edgar2004) with default options. In addition, other taxonomically related species available in GenBank® like Spirocerca lupi, Spirocerca vulpis (Rojas et al. Reference Rojas, Dvir, Farkas, Sarma, Borthakur, Jabbar, Markovics, Otranto and Baneth2018), Thelazia callipaeda (Railliet and Henry 1910), Setaria cervi (Rudolphi 1819), and Gongylonema neoplasicum (Molin 1857) were added to the analysis. Dirofilaria immitis (Leidy 1856) and Brugia timori (Partono 1977) were selected as outgroups. Bayesian inference phylogenetic trees of cox1, ITS1, and 18S data were constructed with the BEAST package 2.5 (Bouckaert et al. Reference Bouckaert, Vaughan, Barido-Sottani, Duchene, Fourment, Gavryushkina, Heled, Jones, Kuhnert, De Maio, Matschiner, Mendes, Muller, Ogilvie, du Plessis, Popinga, Rambaut, Rasmussen, Siveroni, Suchard, Wu, Xie, Zhang, Stadler and Drummond2019). The best nucleotide substitution model for each dataset was selected with JModeltest v2.0 (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012). Tamura-Nei model with gamma distribution and invariant sites was selected for cox1 sequences, Jukes-Cantor model for ITS1 data, and Jukes-Cantor model with gamma distribution for the 18S. Fragments larger than 400 bp were included in the analysis for the ITS1 phylogenetic analysis (i.e. isolates 2, 3 and 10). Furthermore, trees were built with 108 Monte Carlo Markov Chains, 10% burn-in, and sampling every 103 trees. Then, chain convergence and effective sample sizes of each parameter were checked as larger than 300 in Tracer v1.7.2 and trees were summarised in Tree Annotator v2.7.7. Finally, trees were visualised in FigTree v1.4.4. Principal component analysis of cox1 genetic distances was estimated with GenAiEx v6.5 (Peakall and Smouse Reference Peakall and Smouse2012).
Species description
Order: Spirurida Chitwood, 1933
Superfamily: Spiruroidea Oerley, 1885
Family: Spirocercidae Chitwood and Wehr, 1932
Genus: Cylicospirura Vevers, 1922
Cylicospirura wishkai Rojas, Romero-Vega, and Solano-Barquero sp. nov. in Rojas, Romero-Vega, Conejo-Chacón, Hagnauer, Alfaro-Alarcón, Quesada, Alfaro-Segura, and Solano-Barquero, 2025
Description
Zoobank ID: urn:lsid:zoobank.org:pub:CE7F5883-F7A1-40C2-8D66-5B8217766623
Characteristics of adult stage (Figs. 2–4, and Supplementary Figure S1)
Medium-sized elongated spirurids. Red when freshly collected, body cylindrical, and dorsally bent with spiral shape. Cuticle with no ornamentations anterior or posteriorly, transversely striated with 2 μm separation between striae. Hexagonal and sclerotised buccal capsule in face view with rounded vertex (Fig. 3A) armed with six large tricuspid teeth, two lateral, and four submedian. Teeth projected anteriorly with trifurcation at the sixth most anterior end (Figs. 3B and 4A). Accessory teeth absent. Four anterior nipple-like cephalic papillae equally distanced between them. Six bilobated papillae at the interior border of buccal capsule, visible in light microscopy (Figs. 3A and 4B) and SEM (Fig. 3C), each lobe with small opening and aligned with basis of each tooth. Oesophagus divided into anterior cylindrically-shaped muscular portion and a posterior elongated glandular part (Fig. 4A). Oesophageal valve in muscular portion posterior to junction with buccal capsule (Figs. 3B and 4A). Deirids to level of muscular oesophagus. Gut posterior to glandular oesophagus, with an oesophago-intestinal valve. Nerve ring not visible.
Female (based on 10 specimens) (Table 1)
Coiled body 42.043 ± 4.084 mm long and 0.454 ± 0.059 mm wide, with widest portion at the gut level. Buccal capsule 0.121 ± 0.015 mm long and 0.117 ± 0.009 mm wide with six teeth 0.130 ± 0.050 mm long projected to the anterior opening and trifurcated in 0.023 ± 0.010 mm long slits. Oral opening or stoma of 0.089 ± 0.013 mm diameter. Total oesophagus length of 3.661 ± 0.036 mm, with anterior muscular oesophagus smaller (0.409 ± 0.018 mm) than posterior glandular oesophagus (3.372 ± 0.392 mm) with a ratio of 0.122 ± 0.013 and percentage of total oesophagus to total body length of 8.810 ± 1.448%. Uteri didelphic, opisthodelphic. Posterior end with two terminal phasmids and anal opening 0.217 ± 0.055 mm to the tip (Fig. 3D). Vulva anterior to the oesophago-intestinal junction or near it 3.480 ± 0.403 mm from the anterior end (Fig. 3E), salient towards the cuticle. Vagina cylindrical containing numerous eggs measuring 35.8 ± 1.9 μm in length and 15.9 ± 0.9 μm in width (Fig. 4D). Eggs located in uteri and vagina.
Male (based on nine specimens) (Table 1)
Coiled elongated body 29.478 ± 0.738 mm long and 0.298 ± 0.03 mm wide, dorsally curved at the posterior end. Squared buccal capsule 0.105 ± 0.007 wide and 0.105 ± 0.007 long with oral opening or stoma 0.074 ± 0.01 mm in diameter. Teeth 0.111 ± 0.009 mm long emerging from the basal portion of the buccal capsule and projecting anteriorly. Teeth trifurcated distally with 0.016 ± 0.002 mm slits. Oesophagus elongated and cylindrical 2.043 ± 0.69 mm long, with anterior muscular oesophagus 0.329 ± 0.049 mm long, and posterior glandular oesophagus 1.843 ± 0.717 mm long. Muscular to glandular oesophagus ratio of 0.206 ± 0.089 and percentage of total oesophagus to total body length of 7.352 ± 2.538. Testis parallel to intestine in the oesophago-intestine junction. Alae not observed. Posterior end ventrally curved. Transverse striations not visible in the ventral portion of tail and replaced by short longitudinal and parallel cuticular ridges in all area rugosa except surrounding cloaca, subterminal papillae, and gubernaculum (Figs. 3F and 4E). Cloaca slit-like with transversal opening 0.261 ± 0.040 mm to the posterior end. Ten pairs of caudal papillae. Four equidistant, ventrolateral, nipple-shaped and pedunculated pairs of pre-cloacal papillae (Fig. 3G, 3I). Two pairs of nipple-shaped and pedunculated post-cloacal papillae, one pair slightly anterior to the other ventrolateral pair of papillae (Fig. 3G). Four pairs of small, sessile, and subterminal papillae near the distal end. Two elongated and dissimilar spicules emerging from the cloaca. A long and slender major spicule 1.973 ± 0.0016 long with tapered end, and a minor spicule 0.462 ± 0.056 mm long with rounded and hyaline distal end (Fig. 3H). Gubernaculum irregularly-shaped 0.034 ± 0.005 mm to the distal posterior end (Figs. 3H and 4C).
Taxonomic summary
Type specimens: a holotype of an adult male and 18 paratypes of nine males and ten females were deposited in the National Helminthology Collection of Costa Rica, Costa Rica, with catalogue numbers CHCR-213-1 (Holotype, male specimen), CHCR-213-2 (paratype, female specimen), and CHCR-213-3 to CHCR-213-19 (nine males and eight females) as voucher specimens.
Type host: Herpailurus yagouaroundi (Carnivora:Felidae) (Fig. 1), collected in 2019.
Jaguarundi, Herpailurus yagouaroundi, host of Cylicospirura wishkai sp. nov. in the wild; (A) whole body view and (B) head close-up.
Site of infection in host: The nematodes developed within nodules located in the submucosa and mucosa of the gastric wall.
Pathological changes induced in the host: nodular lesions in the submucosa expanded into the overlying mucosa, respecting the lamina propria except at one point, where a fistulizing lesion formed, connecting the nodule contents to the gastric lumen (Fig. 2A). Multiple individuals were often present within a single nodule (Fig. 2B). Nematodes were surrounded by a thick capsule composed of multiple layers of collagen fibres, fibrocytes, and mild neovascularization (Fig. 2C). Moderate numbers of neutrophils and epithelioid macrophages were scattered within the collagenous capsule. Surrounding the nematodes were areas of cellular debris and degenerated neutrophils. The capsule mildly expanded into the muscular layer; however, muscle fibres and serosa remained largely unaffected (Fig. 2C). Transverse sections of the nematodes revealed a distinct coelomic cavity, absence of cuticular ornamentation, coelomyarian polymyarian musculature, prominent lateral cords, and thick-shelled, embryonated oval eggs inside uteri (Fig. 2D) (Gardiner and Poynton Reference Gardiner and Poynton2006). Overall, the lesion caused by this parasite was a pyogranulomatous, multifocal, nodular, and sclerosing gastritis.
Macroscopic appearance and histopathological observations of lesions. A. Exophytic parasitic nodules on the gastric mucosa. B. Dissected parasitic nodule containing numerous adults of Cylicospirura wishkai sp. nov. C. Photomicrograph of a transectional cut of a gastric nodule. D. Transectional section of an adult Cylicospirura wishkai sp. nov.
Light and scanning electron microscopy observations of males and females of Cylicospirura wishkai sp. nov. A. En face view of the hexagonal buccal capsule showing six tricuspid large teeth projected anteriorly (red triangles), two cephalic papillae (black triangles) opposed to each other, and two of the amphids (yellow triangles) (scale bar = 0.060 mm). B. Lateral view of the anterior end of a specimen showing the large elongated trifurcated teeth projected anteriorly (red teeth) and muscular portion of the oesophagus (black triangle) (scale bar = 0.110 mm). Inset image: close-up of trifurcated teeth. C. Close-up of rounded papillae located at internal border of the buccal capsule. D. Posterior end of a female showing the anal opening (black triangle) (scale bar = 0.200 mm). E. Vulvar opening anterior to the esophago-intestinal junction with multiple eggs (Scale bar = 0.100 mm). F. Posterior end of a male, with ventrally curved tip showing four pairs of pre-cloacal papillae and longitudinal parallel short cuticular ridges. G. Posterior end of a male with four pairs of pre-cloacal pedunculated papillae (yellow triangles), two pairs of post-cloacal pedunculated papillae (red triangles), gubernaculum (blue triangle), and the minor spicule (black triangle) (scale bar = 0.250 mm). H. Posterior end of a male specimen showing the minor spicule (black triangle), major spicule (red triangle), and irregularly shaped gubernaculum (yellow triangle) (scale bar = 0.400 mm). I. Close-up of pre-cloacal nipple-shaped papillae.
Line drawings of Cylicospirura wishkai sp. nov. A. Lateral view of the anterior end of a specimen showing terminally trifurcated teeth arising from the third anterior portion of the buccal capsule. The muscular oesophagus is posterior to the buccal capsule, where an esophageal valve is evident. Transverse cuticular striae are represented. B. In face view of the anterior end of a specimen, evidencing a hexagonal buccal capsule with rounded borders, six terminally trifurcated teeth, six bilobulated papillae located in the inner border of the capsula, and six cephalic papillae aligned with base of teeth. C. Lateral view of the posterior end of a male with two unequal spicules, and four pairs of pre-cloacal papillae, two pairs of post-cloacal papillae and an irregularly shaped gubernaculum in the area rugosa. D. Egg with inner embryo or larvae. E. Lateral view of the posterior end of a female showing the annus and a terminal papillae. A version of the uncoloured line drawings is available in Supplementary Figure S1.
Morphometric comparison of Cyclospirura spp. using hierarchical clustering with Ward’s method and radar profiles from standardised characters. A. Hierarchical clustering of female Cylicospirura spp. B. Radar profiles of females considering morphometric values of each colour-coded species. C. Hierarchical clustering of male Cylicospirura spp. D. Radar profiles of females considering morphometric values of each colour-coded species. BCL: buccal capsule length, BCW: buccal capsule width, DVAE: distance of vulva to anterior end, EL: egg length, GEL: glandular oesophagus length, MEL: muscular oesophagus length, MISP: minor spicule, MSP: major spicule, PPOP: pairs of post-cloacal papillae, RGME: ratio of glandular to muscular oesophagus, TBL: total body length, TBW: total body width, TNPPE: total number of papillae in posterior end.
Bayesian Inference phylogenetic analysis of a fragment of the cytochrome oxidase subunit 1 (cox1) of parasites of the families Spirocercidae, Gongylonematidae, Onchocercidae, and Thelaziidae. Posterior probability values (PPV) are next to each node, and the node’s colour and size are proportional to these values. PPV lower than 0.6 are not shown. Parasite family and host family from where the specimens were collected are denoted next to each taxa. Pairwise nucleotide p-distances are denoted as a colour-coded heatmap. Sequences derived from Cylicospirura wishkai sp. nov. are indicated with black diamonds.
Bayesian inference phylogenetic analysis of ribosomal loci ITS1 (A) and 18S (B) fragments. Posterior probability values (PPV) are indicated next to each node, and the node’s size and colour are proportional to these values. PPV lower than 0.6 are not shown. Sequences derived from Cylicospirura wishkai sp. nov. are indicated with black diamonds.
Holotype locality: the holotype specimen was collected in Puntarenas, Costa Rica (8.90152°, –83.11192°).
Paratype localities: Puntarenas, Costa Rica (8.90152°, –83.11192°).
Name of collector: Luis M. Romero-Vega and Isabel Hagnauer.
Prevalence: not calculated
Other material: DNA samples of all specimens are stored in the Laboratory of Helminthology, Faculty of Microbiology, University of Costa Rica.
Representative DNA sequences: representative cox1, ITS1 and 18S sequences were deposited in GenBank® database under accession numbers PV988220-PV988229, PX046119-PX046128, and PV996942-PV996951, respectively (Table 2).
Mitochondrial and ribosomal loci amplified in Cylicospirura wishkai sp. nov. GenBank accession numbers, sequence length, and closest similarity to deposited data
* cox1 accession numbers correspond to concatenated sequences obtained from the two PCRs. Closest GenBank® similarity corresponds to fragment A, obtained with NTF-NTR primers, which have Cylicospirura spp. sequences available for their comparison.
Etymology: the species name is given after the common name of its type host ‘wishka’, which in the indigenous Cabecar language means ‘cat’.
Remarks: Cylicospirura wishkai sp. nov. is macroscopically similar to congeners C. felineus and C. subaequalis, which are also red-coloured nematodes that infect neotropical felids in the Americas and located in stomach nodular lesions. However, these can be distinguished from Cylicospirura wishkai sp. nov. by the following criteria (Table 1): (i) their overall size, a feature that can distinguish the herein described species to C. felineus obtained from Brazil (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020) and Canada (Pence et al. Reference Pence, Samoil and Stone1978) or C. subaequalis from the USA (Waid and Pence Reference Waid and Pence1988); (ii) C. subaequalis has biscuspid teeth, whereas C. wishkai sp. nov. and C. felineus have tricuspid teeth; (iii) vulva of Cylicospirura wishkai sp. nov. is located anterior to the oesophageal-gut junction or near, but in C. felineus the vulva is positioned posterior the oesophageal-gut junction; (iv) glandular and muscular oesophagus of Cylicospirura wishkai sp. nov. males are smaller than C. felineus, but similar to C. subaequalis; (v) Cylicospirura wishkai sp. nov. has four pairs of pre-cloacal papillae and two pairs of post-cloacal papillae, similar to C. subaequalis, but different to the description of C. felineus according to a previous description (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020), but similar to C. felineus described by Pence (Reference Pence, Samoil and Stone1978) and Junker (2024). Therefore, Cylicospirura wishkai sp. nov. can be differentiated from C. subaequalis by its total body length, teeth shape, and number of post-cloacal papillae and distinguished from C. felineus by its total body length, position of the vulva in the female’s body, and the length of the muscular and glandular oesophagus.
Cylicospirura wishkai sp. nov. can be distinguished from other Cylicospirura spp. not reported in the American continent, by: (i) the total body length, being Cylicospirura wishkai sp. nov. considerably longer than other spp., including C. crocutae and C. phiri collected from Crocuta crocuta in Zimbabwe (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024; Junker et al. Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013), C. felinea from Felis silvestris ocreata of Algeria, C. pardalis from Panthera pardus of South Africa (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024), and C. petrowi from Felis chaus in Azerbaijan (Sadykhov Reference Sadykhov1957); (ii) the percentage of oesophagus to total body length was 8.810% in females and 7.352% in males of Cylicospirura wishkai sp. nov. whereas this value was higher than 13% in C. pardalis, C. phiri, C. felinea, C. crocutae, and C. petrowi males, and in females’ data is scarce for those Cylicospirura spp.
Morphological analysis
Sex-specific matrices using only variables with sufficient coverage across taxa were assembled. In addition, species with several missing values (e.g., C. advena) or morphological characters sporadically reported, like dental or valve measurements, were excluded from the clustering (Fig. 5). Statistical differences across species could not be calculated, since other Cylicospirura spp. reports included measurement ranges or means.
In females, the hierarchical clustering (Ward linkage) grouped Cylicospirura wishkai sp. nov. within the C. felineus (sensu Almeida Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020 and Pence et al. Reference Pence, Samoil and Stone1978)-C. felinea cluster, but remained on a distinct branch inside that cluster (Fig. 5A). The radar profile (Fig. 5B) showed a consistent multivariate offset of Cylicospirura wishkai sp. nov. females relative to congeners according to different morphological characters. Cylicospirura wishkai sp. nov. total body length (TBL) of 42.04 mm was within the upper half of the range but accompanied by a relatively small total body width (TBW) of 0.45 mm, close to C. felineus sensu Pence Reference Pence, Samoil and Stone1978 (0.456) and clearly below C. felinea (0.6255), C. pardalis (0.6775), and C. petrowi (0.621), indicating a slenderer habitus. In addition, the buccal capsule length (BCL = 0.112 mm) and width (BCW = 0.117 mm) were intermediate, smaller than C. crocutae (0.168 by 0.136 mm) and much smaller than C. pardalis (0.186 by 0.213 mm). The muscular (MEL = 0.41mm) and glandular (GEL = 3.37) oesophagus of Cylicospirura wishkai sp. nov. were on the lower side relative to most congeners (e.g., C. crocutae with MEL = 0.8175 mm and GEL = 6.3 mm; C. felineus sensu Almeida Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020 MEL = 0.63 mm and GEL = 4.39 mm). Finally, the distance of vulva to anterior end (DVAE = 3.48 mm) of Cylicospirura wishkai sp. nov. was intermediate and egg size modest (length (EL) = 35.8 μm; width (EW) = 15.9 μm) compared with C. advena (52 by 23 μm) and C. petrowi (41 by 22 μm). Overall, female Cylicospirura wishkai sp. nov showed a slender body and comparatively short oesophageal lengths, a combination that sets it off from the C. felineus group despite clustering with it.
Cylicospirura wishkai sp. nov. males formed a cluster near C. petrowi, C. felinea, C. felineus sensu Pence 1988, and C. phiri (Fig. 5C). The radar plot (Fig. 5D) highlighted clear proportional differences in morphological features. For instance, Cylicospirura wishkai sp. nov. had the largest total body length when comparing it to other Cylicospirura spp. (TBL = 29.48 mm), exceeding C. pardalis (25.35 mm) and far above C. phiri and C. subaequalis, while showing the smallest total body width (TBW = 0.298 mm) among reported values. Thus, male Cylicospirura wishkai sp. nov. are conspicuously long and slender, similar to their female counterparts. Moreover, the buccal capsule length and width were among the smallest since several congeners presented wider capsules (e.g., C. felinea of 0.139 mm; C. pardalis of 0.162 mm, C. phiri of 0.146 mm, and C. subaequalis of 0.705 mm). In addition, the MEL (0.329 mm) and GEL (1.843 mm) were smaller than most congeners like C. crocutae and C. felinea and the ratio of glandular to muscular oesophagus (0.179) was relatively high (only CPE is higher at 0.204), indicating a proportionally more developed glandular component relative to the muscular segment in Cylicospirura wishkai sp. nov. Finally, dendrogram analysis showed that minor (MISP = 0.462 mm) and major spicules (MSP = 1.973) were mid-range and the counts of caudal papillae are largely invariant across species and therefore contributed little to discrimination. Together, both sexes place Cylicospirura wishkai sp. nov. close to the C. felineus in tree topology (Fig. 5A and 5C) but exhibit a repeatable multivariate shift characterised by longer and narrower bodies and comparatively smaller oesophageal lengths, with modest buccal capsule dimensions.
Molecular and phylogenetic analyses
Only four sequences of other Cylicospirura spp. are available in GenBank®, i.e. one belonging to C. felineus, one to C. subaequalis, and two to C. petrowi, with overlap to the fragments obtained with NTF-NTR primers. Fragments of 807 bp of the cox1 obtained from Cylicospirura wishkai sp. nov. were 90.01–90.81% similar to Spirocerca lupi collected from a domestic dog from Israel (GenBank® accession number MH633997), whereas fragments obtained with only NTF-NTF primers were 91.15–92.15% similar to Cylicospirura felineus from Lynx rufus of the USA (GQ342967). The phylogenetic analysis revealed two clusters within the Spirocercidae family: one with Spirocerca lupi and Spirocerca vulpis and another one with Cylicospirura wishkai sp. nov., C. felineus, C. subaequalis, and C. petrowi (Fig. 6). Importantly, cox1 sequences derived from Cylicospirura wishkai sp. nov. were differentiated from the three other Cylicospirura spp. available in GenBank® by nucleotide differences of 8.33 to 8.66%. Pairwise p-distances between Cylicospirura wishkai sp. nov. specimens ranged from 0.13 to 1.1%.
Ribosomal DNA loci mirrored the separation of Cylicospirura wishkai sp. nov. from its congeners obtained with cox1 data. Accordingly, ITS1 sequences of Cylicospirura wishkai sp. nov. differed in length due to differences in chromatogram resolution. Seven out of ten sequences were between 210 and 284 bp long, whereas three were 454 and 486 bp long. These ~200 bp differences corresponded to double peaks from position ~240, which may be explained by different ITS1 copies in a single specimen, as observed with Spirocerca spp. (Rojas et al. Reference Rojas, Dvir, Farkas, Sarma, Borthakur, Jabbar, Markovics, Otranto and Baneth2018). Nucleotide p-distance differences between ITS1 sequences of specimens 2, 3, and 10 ranged from 0.3% to 0.9%. Only one ITS1 sequence of Cylicospirura spp., i.e. C. petrowi, was available in GenBank®, and it was distinguished from Cylicospirura wishkai sp. nov. by 15.2 to 15.4% nucleotide differences. ITS1 sequences longer than 400 bp were used in the phylogenetic analysis and showed a separate cluster from C. petrowi, Spirocerca spp., and Gongylonema nepalensis (Fig. 7A); 18S phylogenetic tree divided Cylicospirura wishkai sp. nov. from Spirocerca spp. with high posterior probability (PP), whereas separation between isolates was negligible with PP lower than 0.6. Notably, there was no observed 18S nucleotide divergence among the specimens collected in this study; all exhibited pairwise p-distances of 0 (Fig. 7B).
Discussion
The Spirocercidae family comprises the genera Spirocerca and Cylicospirura, which infect canids and felids, respectively, around the world. Eleven species of Cylicospirura have been reported in wild and domestic felids, two of which have been found in the Americas, namely C. felineus and C. subaequalis (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011). In this study, we describe Cylicospirura wishkai sp. nov. found in the neotropical felid Herpailurus yagouraoundi from Costa Rica. This new species is distinguished from its congeners by differences in mitochondrial and ribosomal gene sequences, as well as by morphological features evident through detailed morphometric analysis.
Jaguarundis (Herpailurus yagouaroundi) are small wild felids distributed from Southern Texas to Northern Argentina. They inhabit a wide range of environments, including tropical forests, savannas, and shrublands, often near water sources. This species is primarily solitary and feeds on small mammals, birds, and reptiles (de Oliveira Reference de Oliveira1998). Although globally listed as Least Concern by the IUCN Red List due to its broad range, populations are declining in many areas as a result of habitat loss, fragmentation, and illegal pet trade (Caso et al. Reference Caso, de Oliveira and Carvajal2015). In Costa Rica, the jaguarundi is classified as endangered under national legislation, reflecting its rarity and vulnerability. Jaguarundis have been previously reported to harbour several helminth parasites such as Physaloptera praeputialis (de Quadros et al. Reference de Quadros, Marques, Moura and Antonelli2014), Echinococcus oligarthrus (Schwantes et al. Reference Schwantes, de Souza Quevedo, D’Avila, de Paula, Fortes and Graichen2021), Taenia omissa (Uribe et al. Reference Uribe, Payan, Brabec, Velez, Taubert, Chaparro-Gutierrez and Hermosilla2021), Aelurostrongylus abstrusus (de Quadros et al. Reference de Quadros, Marques and Ramos2021), C. subaequalis (Vieira et al. Reference Vieira, Muniz-Pereira, Souza-Lima, Marun Rocha and Luque2017), and C. felineus (Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023). In both reports involving Cylicospirura, however, no morphological or molecular data were provided to confirm species identification (Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023; Vieira et al. Reference Vieira, Muniz-Pereira, Souza-Lima, Marun Rocha and Luque2017).
The geographic distribution and the host infection range of Cylicospirura wishkai sp. nov. remain unknown. Given that previous reports of C. felineus and C. subaequalis (Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023; Vieira et al. Reference Vieira, Muniz-Pereira, Souza-Lima, Marun Rocha and Luque2017) in jaguarundis have been based on macroscopic observations of lesions and worms, it is possible that those specimens might correspond to Cylicospirura wishkai sp. nov. A thorough examination of specimens reported by Matos et al. (Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023) and Vieira et al. (Reference Vieira, Muniz-Pereira, Souza-Lima, Marun Rocha and Luque2017) can provide deeper insight into the taxonomy and geographical distribution of Cylicospirura wishkai sp. nov. across the continent. To achieve this, an integrative taxonomic approach is recommended, one that combines morphological, molecular, pathological, and ecological data (Rojas et al. Reference Rojas, Bass, Campos-Camacho, Dittel-Meza, Fonseca, Huang-Qiu, Olivares, Romero-Vega, Villegas-Rojas and Solano-Barquero2025) for every parasitic report.
The comparative morphometric analyses indicate that Cylicospirura wishkai sp. nov. is morphometrically distinct from congeners despite partial clustering with other C. felineus isolates. In females, Cylicospirura wishkai sp. nov. combined moderate to high total body length with relatively low total body width and reduced muscular and glandular oesophageal lengths, producing a compact oesophageal profile not observed in C. crocutae or C. felineus sensu lato (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024; Pence et al. Reference Pence, Samoil and Stone1978). In males, the morphological signal was even clearer since Cylicospirura wishkai sp. nov. exhibited the greatest body length and the smallest body width among the taxa compared, yielding a pronounced elongate, slender complexion. These observations were accompanied by small glandular and muscular oesophagi and a relatively low oesophagus to total body length percentage derived from these structures, supporting a proportionally different oesophageal configuration. Buccal capsule measurements in Cylicospirura wishkai sp. nov. were consistently smaller rather than enlarged, contrasting with species that display broader or longer capsules like C. pardalis, C. felinea, and C. subaequalis (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024; Waid and Pence Reference Waid and Pence1988). Importantly, the traditional diagnostic counts of caudal papillae, including pre-cloacal and post-cloacal, were nearly constant across species and thus are of low discriminant value in this dataset, suggesting the number of these structures is characteristic to the Cylicospirura genus.
Morphometric analysis, incorporating sex-specific dendrograms and standardised radar profiles, consistently showed Cylicospirura wishkai sp. nov. as being distinct from its nearest neighbours in trait space, i.e. C. felineus and C. crocutae for females and C. petrowi and C. felinea for males. The most distinctive morphological characters were: (i) elongate and narrow body proportions, (ii) reduced absolute esophageal lengths with a relatively high glandular/muscular ratio and low percentage of oesophagus to total body length, and (iii) modest buccal capsule dimensions. Our analysis used midpoint values for morphological characters in species where only ranges were reported. This was necessary for several Cylicospirura spp., as available reports did not provide calculated means based on a substantial number of measured individuals (Junker et al. Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013; Sadykhov Reference Sadykhov1957). This in turn prevented our data from using statistical analysis to discriminate among significantly different components. Nevertheless, the sex-parallel multivariate displacement of our specimens relative to its congeners, and the consistent body and oesophagus proportional differences constitute significant quantitative evidence supporting Cylicospirura wishkai sp. nov. as a distinct species. Importantly, this analysis should be interpreted in light of inter-study measurement variance and considering molecular and pathological data.
Analysis of mitochondrial cox1 gene and rDNA ITS1 and 18S loci confirmed the separation of Cylicospirura wishkai sp. nov. from its congeners, i.e. C. petrowi, C. felineus, and C. subaequalis with high genetic divergence (Bisterfeld et al. Reference Bisterfeld, Raulf, Waindok, Springer, Lang, Lierz, Siebert and Strube2024; Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011). Despite the limited number of Cylicospirura spp. sequences available in GenBank® for comparison, our analysis revealed nucleotide differences exceeding 8% in the cox1 gene and 15% in the ITS1 locus between our specimens and its closest relatives. Previous studies indicate that congeneric species typically show cox1 divergence ranging from 6.9 to 16.0% (Blouin Reference Blouin2002), supporting the recognition of Cylicospirura wishkai sp. nov. as a distinct taxon. Analysis of the 18S gene showed the separation of Cylicospirura wishkai sp. nov. from Spirocerca spp. and other filarioids consistent with previous findings (Rojas et al. Reference Rojas, Dvir, Farkas, Sarma, Borthakur, Jabbar, Markovics, Otranto and Baneth2018). This gene is highly conserved and has the least interspecies resolution in the Ascarididae, Ancylostomatidae, and Onchocercidae families, which may also be extrapolated to the Spirocercidae family (Mejías-Alpízar et al. Reference Mejías-Alpízar, Porras-Silesky, Rodríguez, Quesada, Alfaro-Segura, Robleto-Quesada, Gutiérrez and Rojas2024).
Both cox1 and ITS1 markers are recommended for species delimitation and detecting cryptic diversity due to their high interspecies resolution (Chaves-Gonzalez et al. Reference Chaves-Gonzalez, Morales-Calvo, Mora, Solano-Barquero, Verocai and Rojas2022) and broad representation in genetic databases (Mejías-Alpízar et al. Reference Mejías-Alpízar, Porras-Silesky, Rodríguez, Quesada, Alfaro-Segura, Robleto-Quesada, Gutiérrez and Rojas2024). Notably, ITS1 loci tend to accumulate insertions and deletions during DNA replication, making them especially effective for detecting genotypes and discriminating among species (Chan et al. Reference Chan, Chaisiri, Saralamba, Morand and Thaenkham2021). Accordingly, our study observed substantial ITS1 variability among Cylicospirura wishkai sp. nov. specimens, which separated from Gongylonema nepalensis, Spirocerca spp., and Diomedenema dinarctos (of the order Spiruromorpha) collected from crested penguins from New Zealand (Bennett et al. 2022). In addition, unresolved chromatograms appeared in seven out of the ten analysed worms and up to 0.9% nucleotide p-distances were estimated among specimens. This variation probably resulted from the presence of multiple ITS1 copies within a single individual, a phenomenon also observed in S. lupi and S. vulpis and the incomplete rDNA homogenization or concerted evolution (Rojas et al. Reference Rojas, Dvir, Farkas, Sarma, Borthakur, Jabbar, Markovics, Otranto and Baneth2018).
Through both molecular and morphological data, Cylicospirura wishkai sp. nov. was recognised as a separate species; however, the macro and microscopic lesions it caused resembled those produced by other Cylicospirura spp. Nematodes induced nodules in the gastric submucosa, which sometimes connected to the gastric lumen through fistulizing lesions, such as those observed for C. petrowi (Bisterfeld et al. Reference Bisterfeld, Raulf, Waindok, Springer, Lang, Lierz, Siebert and Strube2024), C. subaequalis (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011), or C. felineus (Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023). The nodules from jaguarundis were surrounded by a thick capsule with inflammatory cells, resulting in multifocal, nodular, and sclerosing gastritis as reported for C. felineus in a cougar (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020) and a domestic cat (Crossland et al. Reference Crossland, Hanks, Ferguson, Kent, Sanders and Del Piero2015), and a Cylicospirura sp. from a cat (Ibba et al. Reference Ibba, Lepri, Veronesi, Di Cesare and Paltrinieri2014). Conversely, C. crocutae, C. phiri, C. felinea, or C. pardalis produce nodules in the oesophagus or stomach of their hosts, but no detailed histopathological descriptions are available (Junker et al. Reference Junker, de Klerk-Lorist, Foggin, Martin and Mutafchiev2024; Junker et al. Reference Junker, Lane, McRee, Foggin, van Dyk and Mutafchiev2013). Our results highlight the need of further parasitological analysis when observing nodular lesions compatible with Cylicospirura infections, since the majority of these produce identical manifestations.
The extent and direction of parasite spillover, from wild felids to domesticated ones, warrants particular attention when considering Cylicospirura wishkai sp. nov. and related species. A case of C. felineus was reported in a domestic cat from the USA (Crossland et al. Reference Crossland, Hanks, Ferguson, Kent, Sanders and Del Piero2015), South Africa (Junker et al. Reference Junker, Vorster and Boomker2006), and presumably Italy (Ibba et al. Reference Ibba, Lepri, Veronesi, Di Cesare and Paltrinieri2014). However, these reports lacked molecular data to support the morphological assessment of the parasites, which were identified based on tricuspid teeth in the anterior end (Crossland et al. Reference Crossland, Hanks, Ferguson, Kent, Sanders and Del Piero2015), a characteristic common to other Cylicospirura spp. circulating in the Americas, including C. wishkai sp. nov. This brings profound implications in the control and management of the infection in domestic cats, since the identification of Cylicospirura spp. to the species level can inform about potential wild reservoirs and infection sources. Importantly, jaguarundis have been involved in livestock- and poultry-related conflicts in different regions of the Neotropics, demonstrating their close contact with humans and possibly domestic animals (Giordano Reference Giordano2015).
By employing an integrative taxonomic approach, our hierarchical morphological clustering indicated that Cylicospirura wishkai sp. nov. was most closely related to either C. felineus or C. petrowi, yet it exhibited distinctive differences in body and oesophagus length. Ecologically, C. felineus and C. subaequalis were the anticipated species in the Americas (Almeida et al. Reference Almeida, D’Elia, Sousa, Joaquim, Santos, Campos, Pereira, Soares, Lima and Ecco2020; Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011) and jaguarundis (Matos et al. Reference Matos, Faccin, Dahm, Viott, Walter, Zabott, Pinto, Gruchouskei and Viott2023; Vieira et al. Reference Vieira, Muniz-Pereira, Souza-Lima, Marun Rocha and Luque2017), as earlier reports had documented their presence in these hosts. Histopathological observations, however, revealed that the lesions found in jaguarundis could not be distinguished from those caused by other congeners. When molecular data were incorporated, our specimens showed marked genetic divergence from C. felineus, C. subaequalis, and C. petrowi (Ferguson et al. Reference Ferguson, Woodberry, Gillin, Jackson, Sanders, Madigan, Bildfell and Kent2011), as evidenced by cox1 and ITS1 phylogenetic analysis. Altogether, the evidence provided in this study confirms Cylicospirura wishkai sp. nov. as a new species. Further research is needed to study its prevalence, host range, and other key aspects of its life cycle.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25100977.
Acknowledgements
The authors thank Cristian Fonseca for processing specimens for light microscopy analysis.
Financial support
This project was supported by the project B7733-21 Colección Helmintológica de Costa Rica from the Vicerrectoría de Investigación, University of Costa Rica.
Competing interests
The authors declare no competing interests.
Ethical standard
Animals evaluated in this manuscript were analysed under permit SINAC-ACC-OA-re1389-2022 granted by the Sistema Nacional de Áreas de Conservación de Costa Rica.
