| Chinese alligators(Alligator sinensis) are only distributed in the southern areas of the Yangtze River, yet few reports have ever been on the captive Chinese alligators. So, We make a preliminary report on the intestinal parasites and their diversity in captive Chinese alligators, then identified the advantage nematode Ortleppascaris sinensis according to morphological and molecular characterization, explore its systematic position based on mitochondrial genome and phylogenetic analysis, analyze the damage and then construct a rapid diagnostic methods. We designed to monitor the nematode for breeding Chinese alligator, control the parasitic infection in this endangered creatures. The main results are as follows.1. Diversity of the intestinal parasites in the captive Chinese alligator Alligator sinensisThe present study was conducted at The State of Nature Reserve Chinese alligator in Anhui, Xuanzhou, China. All fecal samples were processed and examined by techniques of physiological saline direct smear, iodine stain, concentrated brine floatation and natural sedimentation, respectively. A total of 328 faecal samples were randomly collected from the captive Chinese alligators. The intestinal parasites finally identified were three protozoa in genus Entaoeba spp., Eimeria spp. and Isospora spp, five nematode in genus Ascaris spp., Ortleppascaris spp., Capillaria spp., Toxocara spp. and Strongylus spp. and one trematode in genus Echinostoma spp., two trematode in family Schistosomatidae and Cryptogonimidae. Interestingly, two or more species of parasite infection were commonly found in one head of a crocodile, and occurrence of parasites was observed in different months or different age of crocodiles. Besides, the number of species, richness index, diversity index and evenness index are higher in July and August, and the juvenile and sub-adults appear mostly affected.2. Morphological and molecular characterization of Ortleppascaris sinensis from the Chinese alligator Alligator sinensisThe specimens of O. sinensis were identified using both morphological and molecular approaches. The morphological characterization of O. sinensis were based on light and scanning electron microscopic examination(SEM), and we also report sequences of the internal transcribed spacers(ITS), small subunit DNA segments(18S) and the cytochrome oxidase I gene(COI). We attribute O. sinensis to the genus Ortleppascaris according to morphological characterization, such as head, oesophagus, stomach, intestine, tail, systema excretorium, copulatory spicules, caudal papillae, gubernaculums, position of vulva, flank and egg, etc. And the sequences obtained from O. sinensis were used in Gen Bank for BLAST search. Unfortunately, there is only one sequence of Ortleppascaris sp. registered in Gen Bank among the genus Ortleppascaris. The maximum score(query coverage, maximum identity) in BLAST analysis was 239(18%, 93%), 750(47%, 98%) and 682(100%, 86%) for ITS, 18 S, and COI sequences, respectively. O. sinensis are from different hosts and territories contrast to other nematodes in genus Ortleppascaris. BLAST analyses supported the morphological identification that O. sinensis sp. nov. is a new biological species. The specific name of this nematode relates to the genitive form of the species name of the host.3. Mitochondrial genome of the nematode Ortleppascaris sinensis and phylogenetic analysis based on AscarididaThis study was under to discuss the systematic position of O. sinensis according to the mitochondrial genome and phylogenetic analysis. We found that the mitochondrial genome of o. sinensis is typical closed double-stranded DNA, 14 328 bp long, and consisted of 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a large non-coding region(AT-rich region), and coded by coding stand. The order of mt DNA gene for O. sinensis is agree with other nematodes of Ascaridida; Most inferred t RNA genes of O. sinensis are 55~63 bp in long, lack stem-loops on either the T-arm or D-arm, and are unable to fold into the t RNA cloverleaf structure; The position of rrn S gene is between trn E and trn S2 UCN, rrn L gene is between trn H and nad3; The position of AT-rich region(452 bp) is between trn S(UCN) and trn N, including several poly T and poly A in initiating terminal and several hypothetical stem-loop structures. According to the mitochondrial sequence of Ascaridida published on Gen Bank, We found O. sinensis independent into a single taxonomic groups based on BI, MP and ML phylogenetic tree. This result indicates that O. sinensis maybe more belong to the family Heterocheilidae.4. Histopathology of gastric wall in Chinese alligator Alligator sinensis infected with Ortleppascaris sinensisThe present study was undertaken to understand the pathopoiesis of O. sinensis through investigating the histopathological alterations of the gastric and intestinal wall from Alligator sinensis infected with such species. Gross appearance of the gastric wall infected with O. sinensis indicated smooth gastric mucosa and regular plica. However, some of the gastric and intestinal walls were found with multifocal ulceration to a certain degree. Further examinations of samples from normal and diseased gastric tissues were executed under the microscope. We found multifocal superficial erosions, mucosa being covered with funnel-like depression in the lesion or some granulomatous inflammation in the gastric and intestinal wall from Chinese alligators infected with nematodes. The inflammatory exudations and sphacelus were filled in superficial erosions. However, the structure of mocosa depression remained intact, though it being thin and sunken. This change was the result of regeneration of the repaired gastric mucosa. The granulomatous inflammation was seen in the mucosa depression region, and located in the submucosa. The histological examination of the stomach revealed presence of superficial ulcer in mucous layer and granulomatous inflammation in submucous layer at entire gastric and intestinal walls of the Alligator sinensis.5. Loop-mediated isothermal amplification(LAMP) assay for the rapid detection of the nematode Ortleppascaris sinensis.For rapid diagnosis the nematode O. sinensis, loop-mediated isothermal amplification(LAMP) was established according to the target sequence 18 S of O. sinensis. The sensitivity of detecting DNA from adult worm O. sinensis based on LAMP assay was 2.75pg/?L, and there were no cross-reaction with Ascaris lumbricoides, Necator americanus, Fasciola hepatica and Schistosoma japonicum. Besides, the specific product based on LAMP was also obtained from the fecal samples of Chinese alligators infected with O. sinensis. This method only needs a water bath pot, using Bst DNA polymerase, under the condition of 65 ?, insulation for one hour, then you can complete the nucleic acid amplification and observe the result through adding acid dye. This method shortens the testing time, benefit to inspection and epidemiological investigation for grass-roots organization and locate. |
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