INTRODUCTION
Tapeworms of the genus Spirometra (Cestoda: Diphyllobothriidae) are intestinal parasites of feline and canine mammals. They have complex life cycles involving 2 intermediate hosts, such as freshwater copepods (Cyclops spp.), followed by amphibians (tadpoles and frogs) amphibians (tadpoles and frogs) or reptiles (snakes) that consume copepods whilst drinking water [1]. The plerocercoid larva, the sparganum, frequently infects humans by the consumption of raw or undercooked meat from the second intermediate or transport host, or consumption of untreated water containing procercoid-infected copepods [2].
Few species of Spirometra have been reported in Africa. S. pretoriensis (=Lühella pretoriense) has been found in the bat-eared fox (Otocyon megalotis) in South Africa (Baer, 1924, 1926) [3,4] and in the African wild dog (Lycaon pictus) in DRC Congo (Baer and Fain, 1955) [5]. S. theileri (=Diphyllobothrium theileri) has been found in the bush cat (Leptailurus serval) and the tiger cat (Felis lybica) near Pretoria, and in the lion (Panthera leo) in DRC Congo (Baer, 1959) [6]. The plerocercoids of Spirometra, known as spargana, have been detected in wild herbivorous mammals such as buffalo (Syncerus caffer), zebra (Equus quagga), wildebeest (Connochaetes taurinus), and warthogs (Phacochoerus africanus) in Tanzania and Kenya [7,8]. Adults of S. theileri were collected from dogs and cats experimentally infected with spargana from warthogs (Phacochoerus africanus) in Tanzania [9]. S. pretoriensis was found in hyena in Ethiopia [10]. The eggs and proglottids of Spirometra had been found in fecal samples of spotted hyenas (Crocuta crocuta) and lions in previous studies [8,11,12]. A high prevalence of Spirometra infection was reported in lions from the Serengeti and Ngorongoro Crater of Tanzania [13]. Spirometra species was commonly found in the intestines of lion and wild dogs in the Luangwa Valley, Zambia [14]. In lions from Tarangire, northern Tanzania, the prevalence of Spirometra was higher than that of other intestinal helminths [15].
Previous studies reported S. ranarum from lions in the Seronera area [16] and S. theileri from leopard (Panthera pardus) and spotted hyena in Maswa Game Reserve [17], which were identified by molecular analysis of mitochondrial cox1 and NADH dehydrogenase subunit 1(nad 1), together with morphological observations. Both of these areas are within the Serengeti ecosystem, which suggested that further studies were needed to clarify the diversity of Spirometra in the various geographical areas of Tanzania.
In this study, we investigated the occurrence of Spirometra in carnivorous mammals (the main definitive hosts for Spirometra) in 2 widely separated and ecologically important areas, the Serengeti ecosystem in northern Tanzania, and the Selous ecosystem in the far eastern part of Tanzania.
MATERIALS AND METHODS
Sample collection and morphological analysis
Tapeworm sample collection was carried out in the Selous and Serengeti ecosystems in Tanzania between 2011 and 2018. These areas were selected owing to its diverse wildlife species. A total of 15 Spirometra samples was collected from the stool and the intestines of the leopard, Panthera pardus, (n=8), spotted hyena, Crocuta crocuta, (n=1), and lion, Panthera leo, (n=6). Of 15 samples, 12 were obtained from Loliondo, Maswa, Tomm camp, and Tototo in the Serengeti ecosystem, and 3 were obtained from Utunge Kingupira in the Selous ecosystem (Fig. 1; Table 1).
The collected worms were preserved separately in 70% ethanol and 10% formalin for molecular and morphological observations. For morphological observations, each gravid proglottid of 15 samples preserved in 10% formalin was gently compressed between 2 glass slides before carmine staining. The morphological characteristics of the samples were compared with those described by Baer 1926 [4] and Faust et al. 1929 [18] for carmine-stained specimens, including the width and length of mature and gravid proglottids, number of uterine loops, position of the uterine pore, genital pore, vaginal opening, size and number of testes, and size of eggs.
PCR and DNA sequencing
Genomic DNA was extracted individually from 15 adult tapeworms using a QIAamp DNA Mini Kit following the manufacturer’s instructions (Qiagen, Valencia, California, USA). A partial sequence of the mitochondrial cox1 gene was employed, as described by Jeon et al. [20]. PCR primers for the amplification of internal transcribed spacer 1 (ITS1) were designed from conserved sequences of the end regions of 18S and 5.8S ribosomal DNA to examine the relationships between congeneric and other closely-related species. Primers used to amplify the ITS1 region were Di-ITS1F (5′-AAC AAG GTT TCC GTA GGT GA-3′) and Di-ITS1R (5′-AGC AGT CTG CGA TTC ACA TT-3′). The 25 μl reaction mixture contained 50 ng of nuclear DNA, 10 pmol of each ITS1 primer, 2.5 μl of 10×buffer, 12.25 μl of 2×buffer (MgCl2, dNTP), and 1.25 units of Taq polymerase (TAKARA Bio, Inc., Kusatsu, Japan). Reaction conditions were 3 min at 95°C, over 35 cycles of 1 min at 95°C, 1 min at 58°C, 1 min at 72°C, and a final extension step of 10 min at 72°C. PCR products were purified and ligated into the pGEM-T vector (Promega, Madison, Wisconsin, USA), transformed into Escherichia coli, and plated onto LB agar containing X-Gal (20 mg/ml) and ampicillin (100 μg/ml). Plasmid DNA from selected colonies was digested by EcoRI to confirm the size of the insert. Ten positive clones from each PCR product were used for nucleotide sequence confirmation.
DNA sequence analysis
DNA sequences of the mitochondrial cox1 gene and ITS1 were assembled using Geneious R9.1 (Biometer, Auckland, New Zealand). The sequences were identified by comparisons with cox1 sequences of Spirometra erinaceieuropaei, S. decipiens, S. ranarum, and S. theileri, and with ITSI sequences of Diphyllobothrium latum and Diphyllobothrium nihonkaiense in the GenBank database.
A phylogenetic analysis was performed by Bayesian inference (BI) and maximum likelihood (ML) using partial mitochondrial cox1 (390 bp) sequences of S. erinaceieuropaei (KJ599680), S. decipiens (KJ599679), S. ranarum (MH298843), D. nihonkaiense (EF420138), and D. latum (DQ985706) and ITS1 (526 bp) sequences of D. nihonkaiense (AB288368 and AB288369). The ML analyses of cox1 and ITS1 were implemented in MEGA7 [19] with the HKY+G substitution model, determined using a Modeltest. BI analyses were performed using Bayesian Evolutionary Analysis Sampling Trees (BEAST) (version 1.10.4) [20,21] with the HKY substitution, which was chosen using MEGA. Nodes were assessed by bootstrapping with 1,000 pseudoreplicates.
RESULTS
Morphological features of Spirometra species in Tanzania
A strobila of S. erinaceieuropaei collected from a leopard measured 45 cm long. The scolex was spoon-shaped, measuring 0.4 mm in diameter and 1.0 mm in length. The proglottid segments measured 5 to 9 mm in width and 5 to 8 mm in length and became broad on the terminal gravid segments. The cirrus pouch appeared just behind the male genital opening close to the vaginal pore, projecting towards the vaginal duct. The uterus covered a narrow field comprising 7 to 8 complete coils, with 4 internal loose coils located slightly behind the outer pile. The eggs were numerous, ellipsoidal, operculated, and up to 57 μm with a diameter of 34 μm, and testes and vitellaria extended to the lateral margin of the uterus and united at the anterior portion (Fig. 2A).
A strobila of S. ranarum collected from the stool of a lion was 75 cm long and 3 mm wide. The mature and gravid proglottids were 12.0 mm in width and 3 mm in length, on average. The uterus comprised 3–3.5 loops with a butterfly or bow-tie appearance, and adopted a diagonal direction in the second turn. The genital and vaginal pores were situated ventrally on the midline in the anterior third of the proglottid. The male genital aperture extended to the anterior border of the segment and the median was close to the female aperture on its lateral side. The testes measured 74 μm in diameter. The ovaries were highly dendritic and connected to the uterus. The eggs were numerous, 57 to 60 μm in length and 34.2 to 35.3 μm in transverse diameter (Fig. 2B; Table 2).
The strobilae of S. theileri collected from the intestine of a leopard and spotted hyena were 45 cm and 134 cm in length, respectively, with varying shapes and sizes. The first segment measured 0.4 mm wide and 0.02 mm long, up to 0.6 to 1.0 mm in width, and 0.15 to 0.25 mm in length. The uterus consisted of 3–4.5 loops on each side of the proglottids. The genital pore was located ventrally on the midline in the anterior 1/5 of the proglottids. The vaginal pore was behind the male genital pore. The uterine pore was situated posterior to the vagina, and numerous thick-shelled and operculated eggs were present, 54 to 59 μm in length and 20 to 24 μm in width. The dumbbell-shaped ovary was located near the posterior margin of the proglottid and connected to the uterus (Fig. 2C; Table 2).
Sequence similarity and phylogenetic relationships
Among the 15 samples, 10 partial cox1 sequences were successfully obtained and deposited in GenBank (under accession numbers MT775875 for S. erinaceieuropaei, MT775874 for S. ranarum, and MT775870-775873, MT775876-775879 for S. theileri). Partial cox1 sequences (−400 bp) from 8 isolates from Tanzania showed 99.5% (400/430) similarity with cox1 of previously sequenced Tanzanian S. theileri (MK955901). Additionally, cox1 of one specimen (No. B473) showed 99.5% similarity with that of Korean S. erinaceieuropaei (KJ599680), and cox1 of another specimen (No. 457-3) showed 99.5% similarity with that of Myanmar S. ranarum (MH298843). Sequence differences in the partial cox1 sequence of Tanzanian Spirometra ranged from 0% to 10.1%. Samples were identified as S. theileri (n=8), S. erinaceieuropaei (n=1), and S. ranarum (n=1) by cox1 sequence analysis. Spirometra theileri, S. erinaceieuropaei and S. ranarum were all found in Serengeti ecosystem while only S. theileri was revealed in Selous ecosystem (Table 1).
The ITS1 sequences of 4 Spirometra species, S. theileri (MN737751, 716 bp), S. erinaceieuropaei (MN733011, 732 bp), S. decipiens (MN733010, 801 bp), and S. ranarum (MN733012, 842 bp), showed length differences. The ITS1 sequence of S. theileri showed 89.8% similarity with that of S. erinaceieuropaei (MN733011), 82.5% similarity with S. decipiens (MN733010), and 78.3% similarity with S. ranarum (MN733012), and 94.4% similarity was observed between S. decipiens and S. ranarum.
Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of. Dibothriocephalus (=Diphyllobothrium).
The cox1 and ITS1, by both ML and BI methods, gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade (Fig. 3). The Dibothriocephalus species were sisters of each other, and, collectively, forming successive outgroups.
DISCUSSION
Spirometra can be identified based on morphological and molecular characters. In this study, S. erinaceieuropaei, S. theileri, and S. ranarum were obtained from lions, spotted hyenas, and leopards in the Selous and Serengeti ecosystems, the 2 most important ecosystems in Tanzania, with a high diversity of wildlife species. Adult Spirometra were analyzed based on genital morphology and mitochondrial cox1 and ITS1 sequences. Partial cox1 sequences obtained from 10 of the 15 samples revealed 3 species of Spirometra were present.
Based on morphological observations, uterine shapes of collected Spirometra species vary with respect to the number of coiled shapes, with 7–8 complete coils in S. erinaceieuropaei, 3–3.5 in S. ranarum, and 3 on one side and 4 complete coils on another side in S. theileri, similar to previous findings [5,22]. The ovaries were clearly dendritic in shape, but the eggs were operculate with slight differences in size among taxa.
Major differences between S. theileri and S. pretoriensis included the number of uterine loops and shape of the cirrus pouch. The uterus of S. theileri consisted of 3–4.5 coils and a cirrus pouch with communication through a short canal with a second smaller vesicular seminis, while the uterus of S. pretoriensis formed a single large loop on either side and the cirrus pouch contained a large vesicular seminis of variable shape. The distinct morphological features of S. ranarum relative to other Spirometra species included posterior uterine coils larger than the terminal uterine ball and coiling of the uteri diagonally rather than spirally. The widths of gravid proglottids and the uterine ball of S. ranarum were larger than those of other Spirometra species, including S. erinaceieuropaei, S. decipiens, S. theileri, and S. pretoriensis.
Mitochondrial cox1 and nad1 sequences of S. ranarum and S. theileri have been reported [16,17]. Our results provide a basis for the accurate molecular identification of Spirometra species from Tanzania by providing sequences for ITS1 and partial cox1 of 10 specimens. The 3 Spirometra species identified have now been found in Tanzania and various Asian countries, including China, Japan, Korea, Laos, and Thailand. The genetic divergence of the partial cox1 sequences among the several species of Spirometra (S. erinaceieuropaei, S. decipiens, S. ranarum, and S. theileri) ranged from 0% to 10.1%. Phylogenetic analyses based on mitochondrial DNA sequences with ML and BI methods showed that S. erinaceieuropaei and S. theileri were separated from other Spirometra species, and S. decipiens and S. ranarum belonged to the same clade.
In addition to Seronera and Maswa, which have been evaluated in previous studies [16,17], our study is reporting 4 new endemic areas of Spirometra species such as Loliondo Game Controlled Area, Tomm camp, and Tototo in the Serengeti ecosystem in northern Tanzania and Utunge-Kingupira in Selous ecosystem in far eastern Tanzania. Among the 15 specimens collected, 13 specimens were identified as S. theileri collected from the lion (n=6) in Maswa area, 3 from the leopard in Utunge-Kingupira area, 2 from the leopard in Tomm camp area and 2 specimens collected from leopard (n=1) and spotted hyena (n=1) in Loliondo area. S. ranarum was obtained from a leopard in the Tototo area and S. erinaceieuropaei was obtained from a leopard in the Loliondo area.
In conclusion, we provide the first evidence that the leopard is a definitive host of S. erinaceieuropaei, S. theileri and S. ranarum and clarified the diversity of Spirometra species in 4 new endemic areas. These findings suggest that wild carnivorous mammals could be as reservoir hosts for Spirometra, which cause human sparganosis in Africa. The infection of wildlife species with Spirometra is likely related to the distribution of Spirometra species in various geographical locations. However, further studies are needed to evaluate the Spirometra species responsible for human sparganosis in the region.
