We discovered that all 3 nose-horned vipers (V. ammodytes) in the Thrace Region of Türkiye were contaminated with the Hepatozoon types. The contaminated vipers were adult women with low parasitemia. We observed fully grown gamonts of Hepatozoon in the erythrocytes of the hosts. Since we did not carry out histopathological evaluations, we might not assess other developmental phases of the parasite. The molecular and phylogenetic analysis confirmed the types as an undescribed Hepatozoon spp. in V. ammodytes.
Species description
Phylum: Apicomplexa Levine, 1970.
Class: Conoidasida Levine, 1988.
Subclass: Coccidia Leucart, 1879.
Order: Eucoccidiorida Leger, 1911.
Suborder: Adeleorina Leger 1911.
Family: Hepatozoidae Wenyon, 1926.
Genus: Hepatozoon Miller, 1908.
Species: Hepatozoon viperoi Ceylan, Ungari and Sevinc sp. nov.
Taxonomic summary
Type-host: nose-horned viper Vipera ammodytes Linnaeus, 1758 (Serpentes: Viperidae: Viperinae).
Type-area: Göztepe area (41° 35′ 3.71″ N, 27° 47′ 48.70″ E) in Vize district of Kırklareli province in Türkiye.
Site of infection: erythrocytes (blood).
Vector: unidentified.
Etymology: thinking about the genus name of the contaminated host (Vipera ammodytes), the protozoan was called Hepatozoon viperoi sp. nov. This is the very first Hepatozoon types reported in the nose-horned viper coming from the Vipera genus.
Parasitemia: 0.1–0.4%.
Deposited products: blood smears and DNA samples from V. ammodytes. These products are transferred in Selcuk University, Faculty of Veterinary Medicine, Department of Veterinary Parasitology.
Gene series: the 18S rRNA concatenated gene series obtained from the blood of V. ammodytes was sent to GenBank under the accession number (OP377741).
Microscopy
We discovered that the nose-horned viper V. ammodytes was contaminated with a Hepatozoon types. In this research study, gamonts of Hepatozoon spp. were spotted microscopically in thin blood smears of all caught horned vipers. Figure 2 programs various tiny pictures of gamonts in each snake, independently. Although the parasitemia level was rather low (< 0.1%) in VA1 and VA3 samples, it was a little greater in VA2 (0.4%) sample. Overall, our morphological and morphometric information together with the molecular and phylogenetic analysis revealed a brand-new Hepatozoon types.
Photomicrographs of intraerythrocytic phases of Hepatozoon viperoi sp. nov. Miller, 1908 discovered in blood samples of Vipera ammodytes; intraerythrocytic gamonts of Hepatozoon viperoi sp. nov. revealing the centric nucleus and no vacuoles [A: VA1 (×100 magnification); B: VA2 (×40 magnification); C: VA3 (×100 magnification)].
Morphological and morphometric analysis of gamonts
Gamonts of the Hepatozoon viperoi sp. nov. were spotted just in the erythrocytes of V. ammodytes, without any other developmental phases being observed in the thin blood smears. Gamonts were confined in a thin parasitophorous vacuole. They were lengthened and had one end more tapered while the cytoplasm was stained whitish pink. Sausage-formed gamonts were discovered near the nucleus of the contaminated erythrocytes, showing a uniform cytoplasm. Furthermore, gamonts displaced the nucleus of the host erythrocytes. Table 2 provides the morphometric criteria consisting of the length and width of the erythrocytes and the length and width of the nucleus of the gamonts. The pinkish-purple gamont nuclei were centrally situated and were mainly quadrangular fit. The length, width, and location of the gamonts (indicate ± basic variance) were 14.05 ± 0.44 µm, 5.83 ± 0.42 µm (n = 15), and 64.96 ± 6.64 µm2, respectively. The length, width, and location of the nucleus of the parasite were 6.35 ± 0.65 µm, 5.20 ± 0.47 µm, and 22.91 ± 3.25 µm2 (n = 20), respectively.
The nucleus of the contaminated erythrocyte had actually an extended look and was compressed in between the gamont and the membrane of the erythrocyte. The contaminated host erythrocytes revealed considerable hypertrophy (P < 0.05) together with greater mean length (P < 0.001) and width (P < 0.05). Although the locations of contaminated erythrocytes were substantially greater than the locations of non-infected erythrocytes (P < 0.001), the location of their nuclei stayed smaller sized (P < 0.05). The length, width, and location of the contaminated erythrocytes were 18.31 ± 0.71 µm, 11.91 ± 0.88 µm (n = 15), and 165.07 ± 11.17 µm2 (n = 20), respectively, while the length, width, and location of the uninfected erythrocytes were 15.60 ± 0.69 µm, 11.22 ± 0.66 µm (n = 15), and 144.83 ± 17.11 µm2 (n = 20), respectively. Table 2 shows the in-depth measurement worths of the morphometric criteria.
Remarks
Although the literature evaluation reported on the existence of Hepatozoon spp. in some snakes of Türkiye, no Hepatozoon types have actually been officially explained (utilizing morphological or a combined technique of morphology and molecular) in snakes up until now. Tome et al. spotted Hepatozoon spp. at the molecular level in some colubrid snakes, consisting of Dolichophis caspius, Elaphe sauromates, and Natrix tessellata, nevertheless, the isolates were not determined at the types level15. This said, no morphological or morphometric information on Hepatozoon types are available in snakes in Türkiye. The morphological and morphometric qualities of Hepatozoon viperoi sp. nov. identified in our research study can be compared to Hepatozoon types discovered in some Brazilian snakes, which might reveal a phylogenetic relationship. To date, although no research studies have actually reported the event of Hepatozoon types in V. ammodytes, some brand-new types consisting of H. cuestensis, H. cevapii, and H. massardi have actually been explained in rattlesnakes (Crotalus durissus terrificus). These rattlesnakes come from the genus Crotalus, subfamily Crotalinae, and family Viperidae. Among these, H. cevapii and H. massardi revealed phylogenetic distance to H. viperoi sp. nov. explained in our research study. Therefore, we utilized these 2 types for the morphological and morphometric contrast. Moreover, we consisted of a just recently explained brand-new types H. annulatum, discovered in a Brazilian colubrid snake Leptodeira annulata, in our contrast due to its phylogenetic distance to H. viperoi sp. nov.
The morphology of gamonts of all previously mentioned Hepatozoon types, consisting of H. viperoi sp. nov., triggered the displacement of erythrocyte nuclei. All types of gamonts seemed lengthened, with one end a little more tapered than the other. The cytoplasmic structures of the H. cevapii and H. massardi gamonts revealed basophilic and granulous cytoplasm. However, the cytoplasm of H. viperoi sp. nov. gamonts revealed harmony. Differentiating H. viperoi sp. nov. from the other types based upon tiny evaluation might be tough due to their comparable morphology. However, at this moment, morphometric measurements can function as a guide.
The length, width, and location of the H. cevapii gamonts discovered in the colubrid snake types Oxyrhopus rhombifer in Brazil were 14.81 ± 0.99 µm, 5.02 ± 0.76 µm, and 64.38 ± 4.70 µm2, respectively, while the length, width, and location of the parasites’ nucleus were 4.35 ± 0.28 µm, 3.99 ± 0.98 µm, and 14.01 ± 1.87 µm2 (n = 30), respectively13. The location of the H. cevapii gamonts resembled that of the H. viperoi sp. nov., with small distinctions in other morphometric measurements. However, considerable morphometric distinctions were observed in between the gamonts’ nuclei. The worths of the nuclei length, width, and location of H. viperoi sp. nov. gamonts were greater than that of H. cevapii.
The length, width, and location of the H. massardi gamonts discovered in Crotalus durissus terrificus types in Brazil were 17.4 ± 0.7 µm, 3.0 ± 0.3 µm, and 38.9 ± 3.7 µm2, respectively, while the length, width, and the location of the parasites’ nucleus were 4.7 ± 0.3 µm, 2.3 ± 0.2 µm, and 9.1 ± 0.8 µm2, respectively9. Although the gamont length of H. viperoi sp. nov. was much shorter than that of H. massardi, the other morphometric measurements consisting of the gamont width and location and the nucleus length, width, and location were greater in the gamonts of H. viperoi sp. nov.
The length, width, and location of H. annulatum gamonts explained in Leptodeira annulata, a colubrid snake types in Brazil, were 14.25 ± 0.54 µm, 5.34 ± 0.26 µm, and 64.32 ± 5.90 µm2, respectively, while the length, width, and the location of the parasites’ nucleus were 3.91 ± 0.63 µm, 4.13 ± 0.29 µm, and 16.95 ± 2.01 µm2, respectively13. The morphometric measurements of the H. viperoi sp. nov. consisting of the gamont’s length, width, and location were discovered to be extremely comparable to that of H. annulatum. However, the measurements of the nucleus of the gamont revealed considerable distinctions in between both types.
Hepatozoon. viperoi sp. nov. spotted in our research study revealed phylogenetic resemblance with H. cevapii, H. massardi, and H. annulatum types discovered in different Brazilian snakes. However, these 3 Hepatozoon types have actually not been spotted in Türkiye yet. The absence of morphological-morphometric studies of Hepatozoon spp. in the snakes of Türkiye led us to compare our findings with those found in the snakes of other countries. Although morphological examination plays a significant role in the differential diagnosis, it is still difficult to predict the Hepatozoon types without morphometric measurements, which requires further molecular confirmation.
Molecular data, sequencing, and phylogenetic analysis
Genomic DNAs were amplified using two sets of primers (HemoF/HemoR, Hep300/Hep900) targeting different regions of the 18S rRNA gene of Hepatozoon. Amplified products were sent for sequencing, and the obtained sequences were submitted to the GenBank database of the National Center for Biotechnology Information. The contig sequences obtained using the pair of primers HemoF and HemoR were further aligned and compared using the Geneious version 7.1.3 (Bomatters, www.geneious.comww)22. Since these sequences were 100% similar, another pair of primers (HepF300/900) was used to amplify another part of the 18S rRNA gene, which was then concatenated with the sequence amplified using HemoF/HemoR resulting in a ~ 1200 bp sequence (OP377741).
Both Bayesian Inference (BI) and the Maximum Likelihood (ML) phylogenetic analyses resulted in identical tree topologies (Fig. 3). The phylogenetic analyses included isolates of adeleorinid parasites (Haemogregarinidae, Hepatozoidae, Karyolysidae, and Dactylosomatidae) available in GenBank. Hepatozoon spp. belong to polyphyletic groups that form two separate clades according to their vertebrate host species. Hepatozoon spp. isolated from large mammals form a sister clade with the Karyolysus genus while the isolates obtained in our study clustered with the sequences of the reptile and anuran hosts belonging to a large Hepatozoidae clade. Although our isolate clustered with the isolates obtained from Brazilian snakes, including Hepatozoon massardi O´Dwyer, Moço, Paduan, Spenassatto, Silva and Ribolla 2013 (KC342526), Hepatozoon cevapii O´Dwyer, Moço, Paduan, Spenassatto, Silva and Ribolla 2013 (KC342525/ON236891) and Hepatozoon annulatum Úngari, Netherlands, Silva and O´Dwyer 2022 (ON262426), it was clustered on a different branch. The gene similarity and pair-wise distance are summarized in Table 3.
Consensus phylogram of haemogregarines based on 18S rDNA sequences. The topology trees with Bayesian inference (BI) and Maximum likelihood (ML) analyses were identical (represented by the BI tree). The values associtaed with the branches are related to the bootstrap values (BI/ML). The scale bar represents 0.02 nucleotide substitutions per site. Adelina dimidiata (DQ096835), Adelina grylli (DQ096836) and Klossia equi (MH211602) were used as outgroups.
