Histomorphological Observation And Population Variation Analysis Of Ascaris Suum In Partial Regions Of Northwest China

Ascaris suum is one of the swine intestinal nematodes, which are widely occurred in both intensive and extensive production systems with a highly prevalent rate throughout the world.Not only can the parasite cause significantly economic losses of pig industry including decreasing the rate of weight gain and feed conversion ratios, and reducing the quality of meat but also it could cause the clinical signs and pathological lesions such as milk spot liver,eosinophilic pneumonia, and immune suppression. More importantly, several studies have demonstrated that A. suum can infect pongo satyrus and human. Therefore, finding effective methods to eradicate A. suum infection is of great significance to the pig industry and human health. In order to control of pig ascariasis effectively, we must identify it accurately. For a long time, the classification of animal parasite is mainly based on morphological characteristics. However, the data of morphology and molecular identification of A. suum have not completed yet. In this study, we have dissected and measured the internal organs of adult A. suum. Using paraffin HE staining techniques, we have observed and described the microstructure of male and female A. suum. At the same time, the mitochondrial genes named cox1, nad1 and nad4 as well as the ribosome gene internal spacer gene(ITS)were sequenced and analysised to research the population genetic variation of A. suum in partial regions of northwest China. The results are as follows:1. The measured result showed that the body length, girth length, esophagus length,spermatheca length, Spermaduct length and testis length of male A. suum were 18.6±2.3 cm,1.2±0.2 cm, 0.6±0.1 cm, 4.9±1.0 cm, 63.8±16.4 cm, 42.6±12.3 cm respectively. While the body length, girth length, esophagus length, Unilateral uterus length, Unilateral oviduct length and Unilateral ovary length of female A. suum were 26.8±2.7 cm, 1.7±0.2 cm, 0.8±0.1 cm,17.7±3.4 cm, 105.6±19.6 cm, 59.0±12.2 cm respectively. After one-way analysis of variance,the male and female A. suum have significant differences in body length, girth length and esophagus length(P<0.01). Using paraffin HE staining techniques, we have obtained the complete organizational chart of A. suum.2. Using the conserved primers NC5/NC2 to amplify the internal transcription spacer(ITS) genes of A. suum(n=23) isolated from different regions in northwest China. After theamplicons were cloned and sequenced, the sequencing results were divided into ITS1, 5.8S and ITS2 according to the public sequence in GenBank. And then those genes were compared and aligned to build phylogenetic tree. The results showed that the ITS genes of all A. suum samples were amplified successfully with the length of 1000 bp approximately, including 450bp-453 bp for ITS1, 159 bp for 5.8S and 272 bp for ITS2 and the intraspecific differences were 0-0.2% for ITS1, 0 for 5.8S, 0 for ITS2. Phylogenetic analysis displayed that all A. suum samples were monophyletic groups and located in different clusters with the reference sequences of the genus of Baylisascaris. Therefore, the ITS genes were not suitable to serve as molecular marker to distinguish the A. suum isolates from different regions in northwest China, but it can be used as intraspecific genetic marker to identify the genus of Ascaris and Baylisascaris.3. A total of 24 Ascaris suum isolated from four regions in northwestern China were studied basing on the sequence of three mitochondrial DNA(mtDNA) regions, namely portions of NADH dehydrogenase subunit 1(pnad1), cytochrome c oxidase subunit 1(pcox1)and NADH dehydrogenase subunit 4(pnad4).Those genes were amplified by PCR method and the lengths of pnad1, pcox1 and pnad4 were 480 bp, 787 bp, and 811 bp respectively. The content of A+T of pnad1, pcox1 and pnad4 gene were 69.69-71.12% 、65.54-65.96% and68.46-69.29% respectively. The intraspecific sequence variations within A. suum samples were 0-2.9% for pnad1, 0-2.1% for pcox1 and 0-3.1% for pnad4. Phylogenetic analysis combined with three sequences of mtDNA fragments showed that Chinese isolates and American isolates might be two different geographical strains and the three mtDNA fragments may be served as genetic marker to distinguish the A. suum samples between China isolates and American isolates, but it could not be used as molecular marker to identify the A. suum isolates from four regions of northwest China.In conclusion, this study not only perfected the morphological data, but also cleared the population differences of A. suum in partial regions of northwest China based on ITS genes and chondriosome genes(nad1, cox1, nad4). These may provide basic data for epidemiological investigation and identification of A. suum.