Observation of sarcocysts in domestic ducks by LM and TEM
The LM examine revealed the presence of sarcocysts in the leg muscle samples from three of the 28 domestic ducks studied (10.7%). The quantity of sarcocysts was comparatively low, and just one to 2 sarcocysts had been discovered in 20 g of muscle tissues from every of these three ducks. All sarcocysts had been morphologically comparable. The sarcocysts had been microscopic, measuring 980–1694 × 72–140 μm (n = 8) in measurement and had a thick striated cyst wall with quite a few brush-like villar protrusions (vps) of 3.8–4.3 μm in size (n = 30) on the cyst floor (Fig. 1a). They had been septate and contained lancet-like bradyzoites of 12.3–14.0 × 1.6–2.3 μm (n = 30) in measurement (Fig. 1b).
The sarcocyst wall contained quite a few lanceolated vps of 2.3–2.5 × 0.3–0.5 μm (n = 10) in measurement. Each vps narrowed in the stalk, widened in the center, tapered on the finish and was lined by a clean electron-dense layer. The bundled microtubules had been current in the core of the vps, which prolonged from suggestions of the vps into the bottom substance. A layer of the bottom substance of 0.3–0.5 μm in thickness (n = 8) was positioned instantly beneath the cyst wall (Fig. 1c, d).
Molecular analyses of sarcocysts in domestic ducks
The three chosen genetic markers (18S rDNA, 28S rDNA and mtcox1) had been efficiently amplified and sequenced. One nucleotide sequence was assembled from three clones of every genetic marker for every duck, and the sequences of every of the three genetic markers had been decided to be 100% an identical among the many three ducks. Therefore, just one sequence of every genetic markers was deposited in GenBank beneath the accession numbers OP480004 for 18S rDNA (1594 bp), OP480005 for 28S rDNA (1558 bp) and OP485287 for mtcox1 (883 bp).
Comparisons of the brand new sequences with these deposited in GenBank confirmed that probably the most comparable sequences had been these of Sarcocystis halieti in the nice cormorant Phalacrocorax carbo and European starling Sturnus vulgaris, and Sarcocystis calchasi in the domestic pigeon (Columba livia) on the 18S rDNA (99.1% id); Sarcocystis wenzeli from the domestic rooster Gallus gallus on the 28S rDNA (95.9–96.0% id); and Sarcocystis speeri from the opossum on the mtcox1 (98.2% id). The new sequences shared not more than 99.0%, 95.6% and 97.7% id on the 18S rDNA, 28S rDNA and mtcox1, respectively, with these of Sarcocystis wobeseri, S. anasi, S. rileyi and S. albifronsi obtained from Anseriformes avian species (Table 1).
Phylogenetic evaluation obtained with the 18S rDNA (Fig. 2a), 28S rDNA (Fig. 2b) and mtcox1 (Fig. 2c) sequences positioned the organism discovered in the domestic ducks inside a clade encompassing Sarcocystis spp. obtained from avian or carnivorous intermediate hosts and avian, marsupial or carnivorous definitive hosts. All three phylogenetic timber had comparable topologies, and the organism described in the current examine shaped a separate department, which didn’t cluster with any of the three foremost clades of Sarcocystis species utilizing birds as intermediate hosts, i.e. S. calchasi, S. halieti and others (chicken–chicken life-cycle), S. rileyi, S. wenzeli and others (birds–placental predatory mammals life-cycle), S. falcatula, S. speeri and others (birds–marsupial/particularly opossum life-cycle).
On the premise of morphological and molecular characterization of the sarcocysts, the isolate from the domestic ducks from China is thought to be a brand new species named Sarcocystis platyrhynchosi n. sp.
Family Sarcocystidae Poche, 1913
Sarcocystis platyrhynchosi n. sp.
Diagnosis: The sarcocysts had been microscopic, as much as 1694 μm lengthy and 140 μm huge. Numerous brush–like vps of 3.8–4.3 μm in size had been current on the cyst floor. TEM statement revealed that the sarcocysts had lanceolated vps of 2.3–2.5 × 0.3–0.5 μm, which narrowed in the stalk. Each vps contained bundled microtubules on the core that penetrated diagonally into the bottom substance.
Type intermediate host: Domestic duck Anas platyrhynchos.
Type locality: Shuangtu (31°15ʹ59ʺN, 108°94ʹ15ʺE, altitude 495 m a.s.l.), Yunyang County, Chongqing City, China.
Site of an infection: Muscular tissues.
Definitive host: Unknown.
Etymology: Latin title of the intermediate hosts is used to call the species.
Molecular characterization: Sequences of the 18S rDNA (OP480004), 28S rDNA (OP480004) and mtcox1 (OP485287) of the brand new species have been deposited in GenBank. At the 28S rDNA and mtcox1 sequences, S. platyrhynchosi is unambiguously differentiated from Sarcocystis spp. obtained from Anseriformes birds.
Deposited specimens: Formalin–fastened tissues containing cysts of S. platyrhynchosi, in addition to photomicrographs from LM and TEM examination of the sarcocysts, have been deposited on the Zoological Specimen Museum of Yunnan University, Kunming, China (assortment quantity Prot202205).
ZooBank registration: To adjust to the rules set out in Article 8.5 of the amended 2012 model of the International Code of Zoological Nomenclature , particulars of the brand new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub: 4FA38B3B-4505-4EF4-AC87-14067AD9FA7A. The LSID for the brand new species title Sarcocystis platyrhynchosi is urn:lsid:zoobank.org:act: D1663E66-23C2-4E50-96C5-9E336318C8EE.
To date, solely 4 named Sarcocystis species (S. rileyi, S. wobeseri, S. anasi and S. albifronsi) and two unnamed Sarcocystis species have been recorded in avian intermediate hosts of the order Anseriformes (Table 2). Among these, sarcocysts of S. rileyi are macroscopic, and people of the remaining 5 species are microscopic. The LM examine revealed that the sarcocysts of S. rileyi, S. wobeseri and Sarcocystis sp. 2 ex Anser caerulescens have skinny and clean partitions, and that these of S. anasi, S. albifronsi and Sarcocystis sp. 1 ex A. caerulescens have thick and striated partitions characterised by radial spines- or finger-like vps on the cyst floor. The unltrastructures of the sarcocysts beforehand described from avian species of Anseriformes are categorized into three TEM wall varieties in response to the classification offered by Dubey et al. . One kind consists of vps with anastomosing branches that’s much like kind 23 in S. rileyi; the second kind has minute undulations on the cyst wall that’s much like kind 1d for S. wobeseri and Sarcocystis sp. 2 ex A. caerulescens; and the third kind has finger-like vps organized in a palisade style that’s much like kind 9a for S. anasi, S. albifronsi and Sarcocystis sp. 1 ex A. caerulescens. In our materials, S. platyrhynchosi sarcocysts had quick, brush–like vps on the cyst floor. The TEM examine revealed that the lanceolated vps had a slender stalk and contained bundled microtubules that penetrated diagonally into the bottom substance; that is roughly much like the TEM wall kind 9d or 11a based mostly on outlines of the lanceolated vps or the options of bundled microtubules prolonged into floor substance, respectively. This TEM wall kind differs remarkably from these of Sarcocystis spp. obtained beforehand from wild mallard ducks and different species of Anseriformes.