Establishment Of A Duplex Nested PCR Method For Isospora Suis And Giardia And The PCR Method Based On Lectin Gene Of Cryptosporidium

Isospora suis is one of the main parasitic in pig farm control.It has a strong pathogenicity to piglets,and often leads to diarrhea and weight loss in piglets.Adult pigs often show recessive infection or carrier,which can pose a potential threat to the health of piglets,and sick piglets can be secondary to other intestinal diseases,resulting in significant economic losses to the affected pig farms.The Giardia is another important intestinal parasite with human zoonosis.The cases of infected pigs have been reported in many countries,indicating that giardia pigs can be used as a source of transmission of Giardia duodenalis infection in human beings.It is of great significance to investigate the infection of Giardia and Isospora suis in pig farms.At present,microscopic morphological detection and single pathogenic PCR detection are commonly used for the examination of these two parasites.The traditional microscope detection method has high requirements for the operator’s technical level and experience,and the positive detection rate is not high;Although PCR detection is highly sensitive,it is time consuming to amplify and detect different gene loci of multiple pathogeny in one sample at the same time.Therefore,a duplex PCR detection method,with high sensitivity to detect two kinds of pathogeny at the same time should be found,which can effectively improve the detection efficiency.Therefore,a highly sensitive and specific duplex nested PCR method was established and optimized for the GDH gene of Giardia duodenalis and the SSU r RNA gene of Isospora suis,respectively.The epidemiological investigation on the infection of Isospora suis and Giardia duodenalis in pig farms in some parts of Henan Province was carried out by using this method,which provided a theoretical basis for understanding the prevalence of Isospora suis and Giardia duodenalis in pig farms.Cryptosporidium is another important zoonotic protozoa that causes gastroe nteritis and acute and chronic diarrhea,Which is widespread worldwide.The e xisting genotyping and subtyping tools of Cryptosporidium have divided it into 36 species and more than 70 genotypes,but still have their limitations.For C.hominis SSU r RNA gene similarity of C.cuniculus,horse genotype,donkey g enotype and from the kangaroo completely similar C.hominis,and the distinction of C.parvum between humans and different animal sources is still difficult to distinguish depend on the type of existing tools.The Lectin gene was used as a genotyping tool,different species of Cryptosporidium were found to have different sequences at this locus,especially different species of Cryptosporidiu m had different numbers of CAA triple codon repeats.According to other prot ozoa related studies,Such as,the effect of Entamoeba histolytica lectin protein on the pathogenicity of amoeba,which is suggested that the protein encoded by this gene affect the invasion,reproduction and pathogenicity of Cryptospori dium.Therefore,the gene locus is likely to be an effective tool for classificati on and genetic study of zoonotic cryptosporidium.1.Establishment and application of a duplex nested PCR method for the detection of Isospora suis and Giardia in pigsIn order to simultaneously detect two important intestinal parasites Isospora suis and Giardia,a duplex nested PCR method was developed and optimized based on GDH gene and SSU r RNA gene respectively.The results showed that the size of products amplified by duplex nested PCR for GDH and SSU r RNA locus were 432 bp and 530 bp,respectively.The sensitivity of the duplex nested PCR to Giardia and Isospora suis was 5.51 pg.L-1 and 385 fg.L-1,showed that the duplex nested PCR had good sensitivity.Clinical test show that 53 clinical samples were tested with optimized duplex nested PCR.The results showed that the positive rate of duplex nested PCR amplification was 20.7%(11/53),the positive rate of Giardia was 5.7%(3/53),and the positive rate of mixed infection was 3.8%(2/53).The results of single pathogen PCR and duplex nested PCR had no statistical significance(P > 0.05).The duplex nested PCR method could be used to detect Giardia and Isospora suis rapidly and accurately.2.Molecular Epidemiological investigation of Isospora suis and Giardia in some areas of Henan ProvinceIn order to understand the molecular prevalence of Isospora suis and Giardia in some areas of Henan Province,212 samples of pig feces were collected.Single nested PCR and duplex nested PCR were used to amplify based on the SSU r RNA gene and GDH gene,respectively.The results showed that the total positive rate of Isospora suis and Giardia was 25.9%(55/212),the total infection rate of Isospora suis was 20.3%(43/212),the total infection rate of Giardia was5.7%(12/212),and the mixed infection rate of the two species was 2.8%(6/212).All the farms were infected with at least one specie of parasite.The infection rate of Isospora suis and Giardia was 35.8%(39/109)in the piglets stage,and the lowest in the fatten pigs stage was 3.4%(1/29).The results showed that the infection rates of Isospora suis and Giardia were different among different age groups.The total positive rate of a pig farm in Zhengyang county was 45.5%(5/11)and the positive rate of a pig farm in Xuchang city was 17.9 %(7/39),which show that the infection of Isospora suis and Giardia is different in different areas.The results show that the infection rate of Isospora suis and Giardia in pig farms in China is higher and the distribution is more extensive,so it is necessary to carry out more in-depth epidemiological investigation on the infection rate of Isospora suis and Giardia in pig farms.To provide a reasonable experimental basis for the prevention and control of porcine isosporosis and giardiosis in pigs.3.Establishment of PCR method for Cryptosporidium identification based on Lectin GeneIn order to understand the sequence difference and genetic evolution of Cryptosporidium Lectin gene in different isolates,the feasibility of Lectin locus as genotyping and phylogenetic analysis of Cryptosporidium was evaluated using SSU r RNA gene as a reference.56 isolates of Cryptosporidium were amplified by nested PCR at Lectin and SSU r RNA locus,respectively.Clustalx was used to compare the amplified sequence with the reference sequence,and the evolution tree was constructed by using the adjacent method(Neighborjoiningmethod NJ)in MEGA 5.The results showed that,Lectin gene locus was used as a typing tool to amplify C.parvum,C.hominis,C.cuniculus and horse genotype,C.hominis from donkey which were very similar to C.hominis from human had successfully amplified the target bands about the size of 450 bp,and then were carried out a phylogenetic tree analysis.Results indicated that,different Cryptosporidium species and the same species derived from different animals were in different branches.Lectin gene locus is very likely to be a new Cryptosporidium classification tool which provides a reference basis for the further development of zoonotic Cryptosporidium classification and genetic research.