Introduction
Broad tapeworms within the genus Spirometra (Cestoda: Diphyllobothriidea) are known to cause sparganosis (i.e. larval infection), a potentially life-threatening zoonotic disease, and spirometrosis (i.e. adult infection), with reports from every continent except Antarctica (Scholz et al., Reference Scholz, Kuchta and Brabec2019; Tran et al., Reference Tran, Tran and Mehanna2019; Kuchta et al., Reference Kuchta, Phillips and Scholz2024). Spirometra species are known to parasitize mammals, amphibians, reptiles, birds and rarely fish (Vettorazzi et al., Reference Vettorazzi, Norbis, Martorelli, García and Rios2023; Kuchta et al., Reference Kuchta, Phillips and Scholz2024). The lifecycle begins when an infected definitive host defecates faeces containing eggs. The egg must reach freshwater where it hatches and releases a coracidium. The lifecycle requires two intermediate hosts, the first intermediate of which is a freshwater cyclopoid copepod which becomes infected upon the ingestion of a coracidium. The coracidium develops into the first metacestode larval stage, known as the procercoid, within the copepod. The second intermediate host, typically an amphibian or reptilebut occasionally mammals such as humans, become infected upon ingestion of the copepod-containing procercoids; however, experimental studies have shown that infection through direct penetration by the procercoid is also possible (Li, Reference Li1929; Mueller, Reference Mueller1938). The procercoid develops into the final metacestode larval stage, the plerocercoid, within the second intermediate host; and this plerocercoid can establish itself in various organs or tissues for several years (Mueller, Reference Mueller1974). Plerocercoids, also termed spargana, can elicit a serious clinical manifestation known as sparganosis within the second intermediate host and a wide range of paratenic hosts. Typically, plerocercoids are located within the subcutaneous tissue, but they can migrate to the viscera, muscles, eyes and central nervous system (Kuchta et al., Reference Kuchta, Kołodziej-Sobocńska, Brabec, Młocicki, Sałamatin and Scholz2021). The definitive host, typically a carnivoran mammal, becomes infected by ingesting tissues of a paratenic or second intermediate host containing the plerocercoid. The plerocercoid may also infect an individual if exposed to an open wound, for instance, in cases where amphibians such as frogs are used as poultices and placed on the wound (Liu et al., Reference Liu, Li, Wang, Zhao and Zhu2015). The plerocercoid develops into an adult cestode within the host’s small intestine and releases eggs into the faeces. In the environment, these eggs embryonate and hatch in freshwater, releasing a coracidium that then completes the life cycle through infection of and development in a freshwater cyclopoid copepod (Mueller, Reference Mueller1974). The prepatent period of this cestode is relatively short with eggs being passed in faeces as early as 2 weeks in cats (Kuchta et al., Reference Kuchta, Phillips and Scholz2024).
Adult Spirometra is generally nonpathogenic within the carnivoran definitive host; however, in some cases, it can cause gastrointestinal disease resulting in vomiting, diarrhoea and weight loss (Conboy, Reference Conboy2009). Infection with the plerocercoid may result in more serious disease and even death depending on the degree of pathology and the tissues involved (Conboy, Reference Conboy2009). In humans, there have been approximately 70 reported cases of sparganosis in North America with the most recent case of a 12-year-old child from Florida in 2024 who presented with a painful subcutaneous mass from which a plerocercoid was surgically excised (Mueller et al., Reference Mueller, Hart and Walsh1963; Taylor, Reference Taylor1976; Griffin et al., Reference Griffin, Tompkins and Ryan1996; Kuchta et al., Reference Kuchta, Scholz, Brabec, Narduzzi-Wicht, Xiao, Ryan and Feng2015; Hawkins et al., Reference Hawkins, Feely, Saulino and Raymond2024). In extreme and rare cases, the plerocercoid asexually reproduces in multiple organs causing a fatal condition known as proliferative sparganosis (Buergelt et al., Reference Buergelt, Ellis and Senior1984; Conboy, Reference Conboy2009; Woldemskel, Reference Woldemskel2014; Tokiwa et al., Reference Tokiwa, Fusimi, Chou, Yoshida, Kinoshita, Hikima, Kikuchi and Ozaki2024).
Advancements in molecular diagnostic methods have contributed to the recent reclassification of Spirometra species; and, while still controversial, species or lineages are generally confined to specific geographic regions. Kuchta et al. (Reference Kuchta, Phillips and Scholz2024) proposed a new classification scheme based on molecular data and geographic location in which there are seven distinct lineages: Spirometra erinaceieuropaei in Europe, Spirometra theileri in Africa, Spirometra asiana in Korea and Japan, Spirometra decipiens in South America, Spirometra sp. 2 in both North and South America, Spirometra sp. 3 in North America, and Spirometra mansoni found worldwide (Yamasaki et al., Reference Yamasaki, Sugiyama, Morishima and Kobayashi2024; Kuchta et al., Reference Kuchta, Phillips and Scholz2024). Prior to this new classification scheme, Spirometra sp. 2 was denoted as S. decipiens complex 1 and Spirometra sp. 3 was within the S. decipiens complex 2. Both species complexes have been molecularly confirmed in North and South America. Kuchta et al. (Reference Kuchta, Phillips and Scholz2024) proposed the S. decipiens complex 2 be divided into North and South American lineages with what is now the free-standing S. decipiens in South America and Spirometra sp. 3 in North America. Additionally, specimens within S. decipiens complex 1 are referred to as Spirometra sp. 2. This newly proposed naming schematic by Kuchta et al. (Reference Kuchta, Phillips and Scholz2024) is used henceforth.
In North America, the first species to be formally described was named Diphyllobothrium (Spirometra) mansonoides, based on specimens from a domestic cat in the northeastern USA (Mueller, Reference Mueller1935). At the time, Spirometra was classified as a subgenus of Diphyllobothrium, but it was later elevated to genus (Mueller, Reference Mueller1937; Mueller, Reference Mueller1938). In fact, the first reports of Spirometra in the USA pre-date the 1935 description of S. mansonoides; in 1927, a cestode was collected from a cat in Louisiana and was morphologically identified as belonging to the genus Dibothriocephalus, previously Diphyllobothrium, subgenus Spirometra. Additionally, tapeworms collected from a cat in Puerto Rico, also in 1927, were classified as Diphyllobothrium mansoni (Dikmans, Reference Dikmans1931). Spirometra mansonoides was originally described based on the morphology of the terminal loops of the uterus. However, this morphological feature is also found within the S. decipiens complex and with no supporting molecular data of S. mansonoides, it is currently not recognized as a valid species (Kuchta et al., Reference Kuchta, Phillips and Scholz2024). Nevertheless, Spirometra eggs have been routinely observed through faecal examinations of dogs and cats mainly from the eastern USA across decades, and often diagnosed either to genus level or as S. mansonoides (Conboy, Reference Conboy2009; Wyrosdick et al., Reference Wyrosdick, Chapman, Martinez and Schaefer2017; Hoggard et al., Reference Hoggard, Jarriel, Bevelock and Verocai2019; Nagamori et al., Reference Nagamori, Payton, Looper, Apple and Johnson2020; Sobotyk et al., Reference Sobotyk, Upton, Lejeune, Nolan, Marsh, Herrin, Borst, Piccione, Zajac, Camp, Pulaski, Starkey, von Simson and Verocai2021). Currently, molecular evidence suggests that the Spirometra species present in the USA include: S. mansoni from a captive Samar cobra (Naja samarensis) in Texas, S. mansoni from an unknown host in Nebraska, Spirometra sp. 2 from a bobcat (Lynx rufus) in Illinois, and Spirometra sp. 3 from captive meerkats (Suricata suricatta) in South Carolina, a black rat snake (Pantherophis obsoletus) in Louisiana, an eastern racer snake (Coluber constrictor), and a western ribbon snake (Thamnophis proximus) both from Mississippi (Jeon et al., Reference Jeon, Park, Lee, Choe, Sohn and Eom2016; Waeschenbach et al., Reference Waeschenbach, Brabec, Scholz, Littlewood and Kuchta2017; McHale et al., Reference McHale, Callahan, Paras, Weber, Kimbrell, Velázquez–Jiménez, McManamon, Howerth and Verocai2020; Yamasaki et al., Reference Yamasaki, Sanpool, Rodpai, Sadaow, Laummaunwai, Un, Thanchomnang, Laymanivong, Aung and Intapan2021; Verocai et al., Reference Verocai, Harvey, Sobotyk, Siu, Kulpa and Connolly2023; Kuchta et al., Reference Kuchta, Phillips and Scholz2024).
This study aimed to elucidate the genetic diversity and potential host associations of Spirometra cestodes in the USA and its territories through molecular analysis of specimens collected from naturally infected animals and submitted to veterinary diagnostic laboratories nationwide.
Materials and methods
Sample acquisition
Samples were requested utilizing the VetPDx (Veterinary Parasitology Diagnostic Network), a listserv founded in 2017 to connect parasitology diagnostic laboratories across North America. A total of 302 samples were received from various locations across the USA, including 25 states and 2 territories (Puerto Rico and the US Virgin Islands). The specimens represented adults (n = 71), plerocercoids (n = 9), and faecal samples from which Spirometra eggs were isolated (n = 222). Of the adult specimens, 16 were collected from domestic dogs (Canis lupus familiaris), 52 from domestic cats (Felis silvestris catus), one from an ocelot (Leopardus pardalis), one from a serval (Leptailurus serval), and one from an unspecified host. The faecal samples consisted of 29 from domestic dogs, 188 from domestic cats, and five from unspecified hosts. Finally, of the plerocercoids, one was obtained from a green iguana (Iguana iguana), one from a rattlesnake of unknown species, one from a wild boar (Sus scrofa), one from an opossum (Didelphis virginiana), one from a White’s tree frog (Litoria caerulea), one from a Cuban knight anole (Anolis equestris), and three from New England cottontail rabbits (Sylvilagus transitionalis). Overall, samples included both fresh and archival samples from teaching and diagnostic laboratory collections in which samples were collected from 1993 to 2024 in various preservatives such as formalin and ethanol.
Sample preparation
The different sample types were prepared as follows for DNA extraction: (1) Faecal samples (n = 222): The faecal samples were processed by double centrifugation sugar flotation using 2 g of faeces (Hoggard et al., Reference Hoggard, Jarriel, Bevelock and Verocai2019; Zajac et al., Reference Zajac, Conboy, Little and Reichard2021). After centrifugation, the top 2 mL of the faecal mixture was siphoned from the 15 mL centrifuge tube and added to a 50 mL conical centrifuge tube. Next, 40 mL of water was added to the tube, vortexed, centrifuged at 400 rcf for 10 min and the supernatant was discarded. This step was repeated. Most of the supernatant was removed, leaving the pellet in approximately 2 mL of water. The pellet and water were then vortexed and strained through a 100 µM ÜberStrainer® (PluriSelect, Leipzig, Germany), followed by straining through a 30 µM ÜberStrainer®. The eggs were then collected from the mesh of the 30 µM ÜberStrainer® and stored in 50 mL conical centrifuge tubes. (2) Adults (n = 71) and plerocercoids (n = 9): Approximately 1–2 cm of each specimen was placed in a 1.5 mL Eppendorf microtube. The preservative in which each specimen had been kept was then evaporated using a Vacufuge® plus centrifuge concentrator (Eppendorf, Hamburg, Germany) for 6 min.
Genomic DNA extraction
Genomic DNA was extracted using different methods and kits for the sample types listed. (1) Eggs: For 68 of the 222 samples from which eggs were isolated, an Omni International Bead Ruptor Elite Bead Mill Homogenizer® (Thermo Fisher Scientific, Waltham, MA, USA) was used to break apart the eggs before DNA extraction. To do this, the faecal sediment was added to a 2 mL tube with 1.4 mm ceramic beads and processed with the following settings: 2-min cycle, 10-sec well, 5.00 m sec−1, and run for 2 cycles. DNA was then extracted from the samples using the Maxwell® RSC Tissue DNA kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The remaining 154 samples containing egg samples were extracted using the Maxwell® RSC Faecal Microbiome DNA kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. (2) Adult worms and plerocercoids: These were initially beaten with beads in a 2 mL tube with 2.8 mm ceramic beads and processed with the following settings: 2-min cycle, 10-sec dwell, 5.65 m sec−1, and run for 2 cycles. DNA was then extracted using the Maxwell® RSC Tissue DNA kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Molecular analysis
All samples were subjected to polymerase chain reaction (PCR) using primers (forward primer PlatCOI F (5ʹ-TTT TTT GGG CAT CCT GAG GTT TAT-3ʹ) and reverse primer PlatCOI R (5ʹ-TAA AGA AAG AAC ATA ATG AAA ATG-3ʹ)) that targeted a portion of the cytochrome c oxidase subunit 1 (cox1) gene in the mitochondrial DNA (mtDNA). A modified protocol from Bowles et al. (Reference Bowles, Blair and McManus1995) was utilized to amplify a fragment of the cox1 gene region in a 25 μL reaction that included Go™ TaqGreen Master Mix (Promega, Madison, WI, USA), 2.5 μL of DNA template and 10 μM of each of primer. Cycling conditions were as follows: denaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 30 sec, 52°C for 1 min, and 72°C for 1 min, followed by a final extension of 72°C for 5 min. DNA extracted from a fragment of an adult Spirometra sp. was used as a positive control, and nuclease-free water was used as the negative control. The PCR products were run on a 1.5% agarose gel and visualized using an ultraviolet transilluminator. The expected size of the amplified products was approximately 400 bp in size. Samples that amplified were purified using the E.Z.N.A.® Cycle Pure Kit (Omega Bio–tek, Norcross, GA, USA) and were sequenced in both directions in a 3730xl DNA Analyser at Eurofins Genomics (Louisville, KY, USA).
Phylogenetic and haplotype analysis
For phylogenetic analysis, sequences were aligned and trimmed using MEGA X version 10.0.5 (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018) and were compared to related sequences from NCBI. The phylogenetic tree was constructed from the generated cox1 gene sequences using the maximum-likelihood method with 1000 bootstrap support and the Tamura-Nei with gamma-distribution (TN93+G) best-fit substitution model (Tamura and Nei, Reference Tamura and Nei1993). Schistocephalus solidus (AP017669) was used as an outgroup.
For the haplotype network analysis, sequences of S. mansoni were analysed. This included 83 sequences (330bp) and 41 reference sequences available in GenBank from Vietnam (n = 1), India (n = 1), Australia (n = 4), Korea (n = 2), China (n = 3), Iran (n = 2), Japan (n = 7), Romania (n = 1), Indonesia (n = 3), New Zealand (n = 1), Colombia (n = 1), Thailand (n = 3), Myanmar (n = 2), Tanzania (n = 1), Cambodia (n = 4), USA (n = 1), and Laos (n = 4). Nine of the S. mansoni sequences generated in our study were removed due to their shorter length. The sequences were prepared in Mega X version 10.0.05 (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018) and DnaSP (v6) (Rozas et al., Reference Rozas, Ferrer-Mata, Sánchez-Del Barrio, Guirao-Rico, Librado, Ramos-Onsins and Sánchez-Gracia2017). The prepared sequences were imported into PopART (v1.7) and the median-joining network method was utilized to construct the haplotype network (Bandelt et al., Reference Bandelt, Forster and Röhl1999).
Results
Of the 302 samples received, quality sequences were obtained from 45% (n = 136) of them and were included in the phylogenetic analysis (Table 1) and submitted to GenBank (Supplementary Table 1). Of these 136 samples, 83.1% (n = 113) came from isolated eggs, 14.7% (n = 20) from adult worms, and 2.2% (n = 3) from plerocercoids. The phylogenetic analysis indicated three distinct clades among the samples that grouped with previously submitted sequences of S. mansoni and Spirometra spp. 2 and 3 (Figure 1). Of the 136 sequences, 67.6% (n = 92) were most similar to S. mansoni, 2.2% (n = 3) were similar to Spirometra sp. 2, and 30.1% (n = 41) were similar to Spirometra sp. 3 (Table 1). Within each sample type, the species breakdown is as follows: (1) Of the 113 egg samples, 67.3% (n = 76) were identified as S. mansoni, Spirometra sp. 3 comprised 32.7% (n = 37), and Spirometra sp. 2 was not identified in any of the egg samples. (2) Of the 20 adult specimens, 65% (n = 13) were identified as S. mansoni, 15% (n = 3) were identified as Spirometra sp. 2, and 20% (n = 4) were identified as Spirometra sp. 3. (3) Of the plerocercoids, 100% (n = 3) were identified as S. mansoni. Regarding host association, samples that grouped with Spirometra sp. 3 were found only in domestic cats, whereas samples grouping with Spirometra sp. 2 were only found in domestic dogs (Table 1). No host association was observed for S. mansoni. Geographically, S. mansoni was identified in 15 different states and Puerto Rico. Whereas, Spirometra sp. 3 was found in 17 different states, and Spirometra sp. 2 was restricted to Florida (Figure 2). Spirometra mansoni and Spirometra sp. 3 had a geographic overlap and were found in 14 of the same states. Additionally, all three lineages were found in Florida.
Figure 1. Maximum-likelihood tree inferred from partial cox1 gene sequences of Spirometra samples from this study and related taxa. Sequences from this study are denoted by a solid black circle (●). The best substitution model used was Tamura-Nei + Gamma distribution. Schistocephalus solidus was used as outgroup. (AUS – Australia; ARG – Argentina; BO – Bolivia; BRA – Brazil; CHL – Chile; CHI – China; COL – Colombia; CR – Costa Rica; ETH – Ethiopia; FIN – Finland; INDO – Indonesia; IND – India; IRA – Iran; KOR – Korea; JPN – Japan; NZE – New Zealand; PR – Puerto Rico; POL – Poland; RUS – Russia; SSU – South Sudan; TZN – Tanzania; UKR – Ukraine; USA – United States of America; VEN – Venezuela; VNM – Vietnam; CT – Connecticut; FL – Florida; GA – Georgia; IL – Illinois; ID – Idaho; IN – Indiana; KS – Kansas; LA – Louisiana; MA – Massachusetts; MD – Maryland; MN – Minnesota; NJ – New Jersey; NH – New Hampshire; NY – New York; PA – Pennsylvania; SC – South Carolina; TN – Tennessee; TX – Texas; WI – Wisconsin).
Figure 2. Distribution of Spirometra species in the USA. (A) Distribution of Spirometra sp. 2. (B) Distribution of Spirometra sp. 3. (C) Distribution of S. mansoni. (D) Historical distribution of molecularly confirmed cases in the USA with pictograms of host species.
Table 1. Geographic distribution and species identity of samples (n = 136) included in the phylogenetic analysis
Percentages are defined as the proportion of samples from a particular state belonging to dog, cat and other* hosts and the Spirometra spp. identified.
* Other includes samples from a serval (Leptailurus serval), a green iguana (Iguana iguana), a White’s tree frog (Litoria caerulea) and a Cuban knight anole (Anolis equestris).
Regarding the haplotype network of S. mansoni, overall, there were 26 different haplotypes identified from the 124 cox1 gene sequences (Figure 3). The network analysis indicated two predominant haplotypes to which most of the samples aligned Haplotype 2 (Hap_2) and Haplotype 4 (Hap_4). Hap_2 had a haplotype frequency of 33.9% (n = 42) among samples, including samples from Connecticut, Florida, Georgia, Indiana, Louisiana, Maryland, New Hampshire, New Jersey, Pennsylvania, and South Carolina and sequences from Oceania (e.g. Australia) and Asia (e.g. China, Japan, Iran and Thailand). Hap_4 had a frequency of 30.6% (n = 38) among samples, including samples from Connecticut, Florida, Illinois, Massachusetts, New Hampshire, Pennsylvania and Texas, and sequences from Oceania (e.g. Australia and New Zealand), Asia (e.g. Japan and Indonesia) and South America (e.g. Colombia). A summary of the geographic origins and haplotype frequencies can be found in Supplementary Table 2.
Figure 3. Median-joining haplotype network of S. mansoni isolates. A total of 124 sequences, 83 from this study and 41 from GenBank, were included for analysis. The size of the circle corresponds to the number of sequences belonging to each haplotype. The network is color-coded to represent the geographical origins of samples within each haplotype.
Discussion
The genus Spirometra is likely the oldest lineage of diphyllobothriidean cestodes; and, since its original description over 200 years ago, the genus has undergone multiple reclassifications (Kuchta et al., Reference Kuchta, Phillips and Scholz2024). Before this study, the molecular characterization of Spirometra isolates from the USA was limited with few studies having been done leading to a significant knowledge gap in species diversity and distribution (Waeschenbach et al., Reference Waeschenbach, Brabec, Scholz, Littlewood and Kuchta2017; McHale et al., Reference McHale, Callahan, Paras, Weber, Kimbrell, Velázquez–Jiménez, McManamon, Howerth and Verocai2020; Kuchta et al., Reference Kuchta, Kołodziej-Sobocńska, Brabec, Młocicki, Sałamatin and Scholz2021; Verocai et al., Reference Verocai, Harvey, Sobotyk, Siu, Kulpa and Connolly2023). Despite S. mansoni being found worldwide, reports of molecular characterization from cases occurring in the Americas have only been recently described (Brabec et al., Reference Brabec, Uribe, Chaparro-Gutíerrez and Hermosilla2022; Verocai et al., Reference Verocai, Harvey, Sobotyk, Siu, Kulpa and Connolly2023; Alvarado-Hidalgo et al., Reference Alvarado-Hidalgo, Campos-Camacho, Arguedas-Morales, Romero-Vega, Alfaro-Alarcón, Anchia-Ureña, Bass, Berrocal-Ávila, Hagnauer, Olivares, Solano-Barquero, Traube-Rivera, Montenegro-Hidalgo and Rojas2024; Wu et al., Reference Wu, Kaneko, Lucio-Forster, Spagnoli, Schultz-Powell, Liotta and Bowman2024); however, it has not been determined if S. mansoni is established and circulating in the Americas, specifically the USA. The first molecular report of S. mansoni in the Americas was from a crab-eating fox (Cerdocyon thous) in 2022 from Colombia (Brabec et al., Reference Brabec, Uribe, Chaparro-Gutíerrez and Hermosilla2022; Uribe et al., Reference Uribe, Brabec, Chaparro–Gutiérrez and Hermosilla2023), followed by a Samar cobra in 2023 from the USA (Verocai et al., Reference Verocai, Harvey, Sobotyk, Siu, Kulpa and Connolly2023). In 2024, there were three published reports that included four dogs, three cats and one coyote (Canis latrans) from Costa Rica (Alvarado-Hidalgo et al., Reference Alvarado-Hidalgo, Campos-Camacho, Arguedas-Morales, Romero-Vega, Alfaro-Alarcón, Anchia-Ureña, Bass, Berrocal-Ávila, Hagnauer, Olivares, Solano-Barquero, Traube-Rivera, Montenegro-Hidalgo and Rojas2024), and two Puerto Rican crested anoles (Anolis cristatellus) from Puerto Rico (Wu et al., Reference Wu, Kaneko, Lucio-Forster, Spagnoli, Schultz-Powell, Liotta and Bowman2024). These cases confirm the presence of S. mansoni in the Americas. In the case of the Samar cobra from the USA, which was imported from the Philippines, it was suspected that the snake was infected prior to importation to the USA (Verocai et al., Reference Verocai, Harvey, Sobotyk, Siu, Kulpa and Connolly2023). Additionally, another specimen from an unknown host in Nebraska, USA that was originally labelled as Spirometra mansonoides based on morphology was molecularly misidentified as S. decipiens and later reclassified as S. mansoni (Jeon et al., Reference Jeon, Park, Lee, Choe, Sohn and Eom2016; Yamasaki et al., Reference Yamasaki, Sanpool, Rodpai, Sadaow, Laummaunwai, Un, Thanchomnang, Laymanivong, Aung and Intapan2021, Reference Yamasaki, Sugiyama, Morishima and Kobayashi2024). The results now confirm that S. mansoni is likely well established, across the USA, using dogs and cats as definitive hosts, and has had multiple introductions over time, followed by some events of within-country expansion through animal movement. The latter claim is supported by the results of the haplotype network analysis, which indicates intraspecific genetic diversity within S. mansoni (Figure 3). While various haplotypes comprised sequences from the USA and other continents, their specific origin cannot be determined.
Regarding Spirometra sp. 2, previously named S. decipiens complex 1, there has been only a single molecular report from the USA in a bobcat (L. rufus), whereas all other reports have originated primarily from both wild and domestic canid and felid hosts in South America (Petrigh et al., Reference Petrigh, Scioscia, Denegri and Fugassa2015; Almeida et al., Reference Almeida, Coscarelli, Melo, Melo and Pinto2016; Waeschenbach et al., Reference Waeschenbach, Brabec, Scholz, Littlewood and Kuchta2017; Arrabal et al., Reference Arrabal, Pérez, Arce and Kamenetsky2020; Fredes et al., Reference Fredes, Mercado, Salas, Sugiyama, Kobayashi and Yamasaki2022; Kuchta et al., Reference Kuchta, Phillips and Scholz2024). In contrast, samples from this study included only three cases that grouped with Spirometra sp. 2, all originating from domestic dogs from Florida (Figure 1). It is plausible that the importation of domestic dogs and cats from South America has led to the establishment of Spirometra sp. 2 in North America as well (Jeon et al., Reference Jeon, Park, Lee, Choe, Sohn and Eom2016). However, Spirometra has been reported from a grey fox (Urocyon cinereoargenteus) and Florida panthers (Puma concolor coryi) (Conti, Reference Conti1984; Foster et al., Reference Foster, Cunningham, Kinsella, McLaughlin and Forrester2006), bobcats from Arkansas (Heidt et al., Reference Heidt, Rucker, Kennedy and Baeyena1988), and coyotes and raccoons (Procyon lotor) from various states (Harkema and Miller, Reference Harkema and Miller1964; Schaffer et al., Reference Schaffer, Davidson, Nettles and Rollor III1981; Gompper et al., Reference Gompper, Goodman, Kays, Ray and Fiorello2003) without molecular characterization; therefore, it is plausible that Spirometra sp. 2 was already established and circulating within the USA. Considering the variety of both wildlife and domestic animal host species that Spirometra sp. 2 infects across North and South America, determining any host association within this complex remains challenging. This highlights the need for broader sampling and molecular characterization of specimens from the USA, as well as Central and South America, to better understand the species composition and host associations among different genetic lineages.
Molecular confirmation of Spirometra sp. 3, previously within S. decipiens complex 2, infections have been reported prior to this study in the USA in reptiles and exotic mammals (Waeschenbach et al., Reference Waeschenbach, Brabec, Scholz, Littlewood and Kuchta2017; McHale et al., Reference McHale, Callahan, Paras, Weber, Kimbrell, Velázquez–Jiménez, McManamon, Howerth and Verocai2020; Kuchta et al., Reference Kuchta, Phillips and Scholz2024). In South America, S. decipiens, previously within S. decipiens complex 2, has been found in a maned wolf (Chrysocyon brachyurus) in Bolivia, a Patagonian green racer (Philodryas patagoniensis) in Uruguay, and a Geoffroy’s cat (Leopardus geoffroyi) and an ocelot (Leopardus pardalis) in Brazil (Kuchta et al., Reference Kuchta, Phillips and Scholz2024). The samples that grouped with Spirometra sp. 3 were exclusively detected in domestic cats, suggesting a potential host association within the USA. The sequences from this study represent the first molecular characterization of Spirometra isolates from the geographic region in which S. mansonoides was first described by Mueller (Reference Mueller1935), supporting the idea that S. mansonoides belongs in the Spirometra sp. 3 Complex. According to Kuchta et al. (Reference Kuchta, Phillips and Scholz2024), isolates within S. decipiens complex 2, should be separated into a North and a South American lineage, with those from South America being referred to as S. decipiens and those from North America as Spirometra sp. 3. The results of this study further support the hypothesis of S. mansonoides to be a valid species, possibly represented by Spirometra sp. 3 (i.e., Kuchta et al., Reference Kuchta, Phillips and Scholz2024). Nevertheless, integrated molecular and morphological evidence would be required for resurrecting the species. This could be accomplished by reassessing the type–material of the original S. mansonoides description by Mueller (Reference Mueller1935), in addition to an attempt of molecular characterization of the material, which may not be fruitful given preservation methods, DNA quality, and destructive use. Alternatively, adult specimens could be collected from the type–host and the type–locality (i.e. Syracuse, New York) to provide a robust molecular characterization followed by phylogenetic analysis for comparison with the data generated by our study.
There are many challenges associated with determining a species based solely on morphology. Historically, the reproductive system of adults was used for identification, specifically the number of uterine coils; however, this can vary as the worm develops and is considered unreliable (Iwata, Reference Iwata1972; Yamasaki et al., Reference Yamasaki, Sanpool, Rodpai, Sadaow, Laummaunwai, Un, Thanchomnang, Laymanivong, Aung and Intapan2021). Additionally, the morphology of museum or archival specimens may be distorted, depending on the fixation technique used, and result in misidentification. Furthermore, the opportunistic nature of our sampling limited the ability for morphology to be assessed as the amount of specimen provided was enough only for molecular analysis. Another challenge when working with archival samples, such as the ones obtained in this study, that had an unknown preservation history, is that preservatives such as formalin can greatly inhibit molecular analysis (Zimmermann et al., Reference Zimmermann, Hajibabaei, Blackburn, Hanken, Cantin, Posfai and Evans2008). Furthermore, most samples included in our study were eggs isolated from faeces, a biological sample known to contain PCR inhibitors such as complex polysaccharides, bilirubin, and metabolites from digestion that may have contributed to the reduced number of high-quality sequences suitable for inclusion in the phylogenetic analysis (Roncancio-Duque et al., Reference Roncancio-Duque, García-Ariza, Rivera-Franco, Gonzalez-Ríos and López-Alvarez2024). To have a complete taxonomic description, future studies should focus on both molecular and morphologic characterization of larval and adult stages using standardized fixation techniques that are compatible with molecular techniques, (Chávez-González et al., Reference Chávez-González, Morales-Calvo, Mora, Solano-Barquero, Verocai and Rojas2022; Chervy, Reference Chervy2024).
This study identified patent infections in both dogs and cats, which may increase the risk of transmission in companion animals and pose an additional threat to public health due to the zoonotic potential of this parasite. Infections with Spirometra adults in the small intestine of cats and dogs are typically subclinical, but there are rare instances in which a fatal proliferative sparganosis can occur in companion animals and humans. Proliferative sparganosis develops when the plerocercoids asexually reproduce and migrate into multiple tissues and organs as opposed to non-proliferative infections in which only a few plerocercoids migrate in a confined area within connective tissue (Kikuchi and Maruyama, Reference Kikuchi and Maruyama2020). Historically, reported cases of symptomatic or fatal proliferative sparganosis in humans or animals, thought to be caused almost exclusively by an isolate phylogenetically closely related to Spirometra isolates from South America belonging to Spirometra species 2, therefore within the S. decipiens complex 1, incorrectly referred to as Sparganum proliferum (Miyadera et al., Reference Miyadera, Kokaze, Kuramochi, Kita, Machinami, Noya, Alarcón de Noya, Okamoto and Kojima2001; Kikuchi and Maruyama, Reference Kikuchi and Maruyama2020; Kikuchi et al., Reference Kikuchi, Dayi, Hunt, Ishiwata, Toyoda, Kounosu, Sun, Maeda, Kondo, de Noya, Noya, Kojima, Kuramochi and Maruyama2021; Fredes et al., Reference Fredes, Mercado, Salas, Sugiyama, Kobayashi and Yamasaki2022); however, a recent case of proliferative sparganosis in a cat in Japan was due to S. mansoni (Tokiwa et al., Reference Tokiwa, Fusimi, Chou, Yoshida, Kinoshita, Hikima, Kikuchi and Ozaki2024). The Japanese case highlights the importance of determining the taxonomic status and distribution of Spirometra, as different species may be associated with a higher degree of pathogenicity. Furthermore, feral cats and other wild carnivoran mammals such as raccoons, may act as reservoirs for Spirometra species and potentiate their spread to vulnerable or endangered species in zoological facilities, as was demonstrated by McHale et al. (Reference McHale, Callahan, Paras, Weber, Kimbrell, Velázquez–Jiménez, McManamon, Howerth and Verocai2020) in which three cases of sparganosis in captive meerkats at a zoological facility was reported.
Although Spirometra infections have been reported in Oklahoma, Hawaii, North Carolina and West Virginia, no samples from these areas were available for inclusion in the analysis (Nagamori et al., Reference Nagamori, Payton, Looper, Apple and Johnson2020). Due to the opportunistic and voluntary nature of the sampling strategy employed in the present study, we were unable to confirm the absence of Spirometra spp. in states without sample representation. Notwithstanding the limitation of sampling bias, the results still demonstrated an extensive geographic overlap of S. mansoni and Spirometra sp. 3 with both in 14 of the same states (Figure 2). It is plausible that cats in these states are at an increased risk for co-infection with both lineages. These findings raise the question of how both lineages became established in the same geographic area in the USA. To answer this, we must consider the origins of both the parasite and its host. Spirometra isolates within Spirometra sp. 3 may have already been present in North America with the bobcat (L. rufus), a felid native to North America, serving as the definitive host, and native amphibian and reptile species acting as the second intermediate host (Mueller, Reference Mueller1974). With the colonization of North America by European settlers in the early 16th century, various domestic animals were introduced, including domestic cats. It can be speculated that these animals preyed upon the second intermediate hosts, which led to their infection and integration into the Spirometra life cycle (Serpell, Reference Serpell, Turner and Bateson2014).
In contrast, the results indicate that S. mansoni is currently well established throughout the USA, and while it is speculated that S. mansoni currently has a cosmopolitan distribution with potential origins in Asia, prior to this study there was little molecular evidence to confirm its presence and establishment in North America. Our haplotype analysis further supports the hypothesis of multiple introduction events throughout modern times resulted in the establishment and expansion in the USA. Another explanation, especially pertinent in the last few decades, is the increased travel and rehoming efforts of companion animals such as dogs and cats both domestically and internationally may have further contributed to the introduction of S. mansoni into new geographic areas within the USA (Wright et al., Reference Wright, Jongejan, Marcondes, Peregrine, Baneth, Bourdeau, Bowman, Breitschwerdt, Capelli, Cardoso, Dantas-Torres, Day, Dobler, Ferrer, Gradoni, Irwin, Kempf, Kohn, Krämer, Lappin, Madder, Maggi, Maia, Miró, Naucke, Oliva, Otranto, Pennisi, Penzhorn, Pfeffer, Roura, Sainz, Shin, Solano-Gallego, Straubinger, Tasker, Traub and Little2020; Giannelli et al., Reference Giannelli, Schnyder, Wright and Charlier2024). Additionally, the legal and illegal importation of exotic animals to the USA may play an extensive role in modern times in the introduction and spread of zoonotic pathogens such as species of Spirometra (Rush et al., Reference Rush, Dale and Aguirre2021, Verocai et al., Reference Verocai, Harvey, Sobotyk, Siu, Kulpa and Connolly2023).
Regarding treatment and prevention, there are currently no FDA-approved products labelled for the treatment of Spirometra in companion animals. However, treatment recommendations consist of oral or subcutaneous administration of praziquantel at 25 mg/kg once a day for two consecutive days for both cats and dogs (Conboy, Reference Conboy2009). In cases of proliferative sparganosis, one of the only known successful treatments was of a dog, administered 3 weeks of mebendazole at 20 mg/kg orally once a day followed by 3 weeks of praziquantel at 5 mg/kg orally or subcutaneously once a day, and alternating this regimen for 3 months (Beveridge et al., Reference Beveridge, Friend and Jeganathan1998). Effective prevention strategies must consider the various transmission routes the parasite utilizes to complete the life cycle. A vertebrate host may become infected by (1) ingestion of an infected copepod first intermediate host with a procercoid through contaminated water or food; (ii) ingestion of a second intermediate or paratenic host infected with a plerocercoid; or (iii) migration of a plerocercoid into an open wound of a potential host (Mueller, Reference Mueller1974; Li et al., Reference Li, Song, Li, Lin, Xie, Lin and Zhu2011). Infections in humans primarily occur due to poor food safety and hygiene practices, such as the ingestion of raw or undercooked second intermediate or paratenic hosts, or the use of amphibian poultices on open wounds to facilitate healing in certain cultures (Li et al., Reference Li, Song, Li, Lin, Xie, Lin and Zhu2011; Liu et al., Reference Liu, Li, Wang, Zhao and Zhu2015). Education is likely the most vital tool in prevention of human sparganosis and should focus on educating people about how infections occur, emphasizing food safety (i.e. thoroughly cooking meat, especially wildlife), and filtering potentially contaminated drinking water. In regions where the use of frog and snake poultices is common, discouraging this practice and educating about the risk of infection is warranted (Li et al., Reference Li, Song, Li, Lin, Xie, Lin and Zhu2011; Liu et al., Reference Liu, Li, Wang, Zhao and Zhu2015). In companion animals, prevention could consist of restricting pets’ access to areas which wildlife inhabit and may contaminate the environment, and monitoring pets to prevent ingestion of potential second intermediate or paratenic hosts. Zoological facilities should consider similar mitigation strategies by implementing surveillance of potential reservoir hosts such as feral cats and raccoons that may contribute to contaminating the environment (McHale et al., Reference McHale, Callahan, Paras, Weber, Kimbrell, Velázquez–Jiménez, McManamon, Howerth and Verocai2020).
Conclusion
Spirometra mansoni is well established in the USA and likely has had multiple introductions throughout history. There are two other distinct species of Spirometra present within the United States that correspond to Spirometra species 2 and 3. Two lineages within Spirometra sp. 3 should be considered, one which represents the North American lineage and the other the South American lineage of Spirometra. Pending additional integrated classical and molecular assessment, S. mansonoides may be resurrected as the taxon shown to infect domestic cats in North America. Overall, this study reinforces the need for further molecular characterization of different Spirometra life stages in domestic animals, animals housed in zoological facilities, and wildlife.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S003118202500054X.
Data availability statement
Data supporting the conclusions of this study are included in the article. Generated sequences were submitted to GenBank database under accession numbers: PQ673870 – PQ674005.
Acknowledgements
We would like to thank all those who contributed with samples; in particular, Shane Azumally, Christina Lara Guerra, Dana Brooks, and Hailey Jung at Antech®, and Fiona Pelah at VCA Animal Hospitals™ for coordinating the shipment of samples. We also thank Faith Lass at Texas A&M University for assistance in sample processing. This project would not have been possible without their collaboration and participation.
Author contributions
The study was conceived and designed by C.S., G.V. and M.L. Samples were provided by C.S., P.C., A.V., A.L., A.A., B.D., G.M., C.L., C.L., J.G., H.W., J.B., J.S., C.P., S.D., M.B., M.L. and G.V. Sample preparation and molecular analysis were carried out by T.S. The paper was written by T.S., C.S., G.V., P.D.J.C. and M.L. All authors have read and agreed to the present version of the manuscript.
Financial support
This research received no specific grant from any funding agency, commercial, or not-for-profit sectors.
Competing interests
The authors have no conflicts of interest to declare.
Ethical standards
Not applicable.
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