Genetic Evolution Of Bacillus Subtilis And Establishment Of Nested PCR Method

Besnoitia besnoiti mainly parasitize in the scleral conjunctiva, skin, subcutaneousconnective tissue, mucosal membranes, testes, vagina of Bovine, and result in emaciation,anemia, swelling, digestive dysfunction, which may cause cattle to miscarry, reduce milkproduction, decline seminal quality, and even lead to death. Therefore, Besnoitiosis brings to thecattle industry of great economic losses.In recent years, researches on B. besnoiti have mainly focused on the epidemiology,diagnosis, treatment, and prevention. But at molecular level, there are only the part experimentalstudies about internal transcribed spacer (ITS) of ribosomal DNA, and the completedmitochondrial genome has not been reported. So first obsject of this study was to obtain thecompleted mitochondrial (mt) genome and ITS-1sequence of B. besnoiti in China using themethods of molecular biology, and then their nucleotide sequences were sequenced and analyzed.And phylogenetic trees were constructed based on the protein-coding genes of mitochondrialgenome and the ITS-1to analyze the evolutionary relationships and position of B. besnoitiamong apicomplexan parasites. The results showed that the complete rmitochondrial genomewere3679bp in length, which included the2proteins genes (cytb and cox1), and7transferRNA genes. The nucleotide compositions of the mt genome included29.14%,19.30%,18.48%and33.08%of A, C, G, T, and62.22%for A+T contents and37.78%for G+C contents,respectively. The mitochondrial genome of B. besnoiti was a clear preference for bases A and T.Phylogenetic relationships were constructed based on2proteins genes of the B. besnoitimitochondrial genome, which the results were similar using maximum parsimony (MP),maximum likelihood (ML) and Bayesian (BI) three methods. The results showed that the treedivided into two major cbranches, including Lobosia and Eucoccida branches. On the Cucoccida,Eucoccida and Piroplasmida were in different clade. On the Eucoccida subclass, Haemosporinaand Eimeriina respectively located distinct evolutionary line. On the Eimeriina subclass,Eimeriae was one big branch, while other Family, shch as Cryptosporididae and Sarcocystidaeand Toxoplasmatidae were in another clade. And B. besnoiti, Toxoplasma gondii, Neosporacaninum and Hammondia heydorni were in one sister clade, which had close relationships. Theseresults were consistent with those of previously morphological classification, suggesting that the complete mt genome sequence was a reliable genetic marker for phylogenetic studies of parasite.Based on the ITS-1sequence, this study also utilized MP, ML and BI three methods to constructphylogenetic tree, which B. besnoiti, Toxoplasma gondii, Neospora caninum and Hammondiaheydorni were in one sister clade. And the results were similar with that based on mitochondrialgenome. Also, the results showed that the B. besnoiti China isolate was same as the B. besnoiti inAmerica, Germany,Australia, Israel and so on.At present, diagnostic method of Besnoitiosis has still been far too dependent on the clinicalobservation, while infectious cattle have no obvious clinical symptoms until the very late stagesof disease. At this time, infectious cattle have to be flushed out, and also it can cause largeeconomic loss to livestock. So this study was to establish a rapid and accurate diagnosis methodat the molecular level, which could reduce the economic loss. According to the published B.besnoiti ITS-1sequence in GenBank, two pairs of specific primers were designed to establish adetection method of nested PCR. Genomic total DNA was extracted from infectious cattle wholeblood, and ITS-1sequence was amplified by nested PCR and sequenced for comparativeanalysis, which has been accomplished mostly by trail-and-error until a rapid and specificdiagnosis method was established. Then,196serum samples from the western region ofHeilongjiang Province were tested using the method and the positive rate1.53%.In this study, the complete mt genome sequences of Besnoitia besnoiti in China were firstlyobtained, and analysis and research on their characteristics were performed, which these resultsenriched information of protozoon parasites mt genome dataset. Phylogenetic trees based onribosomal DNA sequences and mt genome sequences clearly indicated genetic relationships of B.besnoiti and other protozoon parasites, and laid a foundation for further study of populationgenetics, molecular taxonomy and phylogenetics of Besnoitia sp.. And a detecting early bovinebesnoitiosisis Nested PCR method were firstly established, and apply this method to investigateB. besnoiti infection status in the part region of Heilongjiang Province, which the results providea reliable base data for further prevention of the Bovine besnoitiosis.