The Polymorphism Of The CoxⅠ Gene Of Trichinella And Quarantine Of Pre-encapsulated Larva In Meat

Trichinellosis is a serious food-borne parasitic zoonosis,the disease was acquired by ingesting the raw or insufficiently cooked meat containing the Trichinella larvae.Because there are distinct differences among different species.Such as,geography distributing,the range of host,the infective ability to host,immunity response of host to different species.So, it is very necessary to study on the classification of Trichinella genus.In addition,along with development of molecular biology,research on the Trichienlla genus will provide the new evidence for Trichinella aetiology,immunology,epidemiology,clinic,prevention and control. Trichinellosis has not only endanger human health seriously,but also has brought great loss to people in pig feeding and food industry.EU law requires that all pigs raised in 15 countries must be examined for Trichinella,which means that the EU spends about US$ 570 000 000 per year for the control of this zoonosis in the domestic pig population.In China,Trichinella is a compulsive item to quarantine in meat,the expenses for quarantine reach to 1 800 000 000 RMB annually.In order to identify the different Trichinella isolates from China at molecular level and analyze their polymorphism,polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and PCR-single-strand conformation polymorphism(PCR-SSCP) were used to observe the polymorphism of the partial mitochondrial cytochrome c-oxidase subunitⅠ(COXⅠ) gene of seven isolates from China in this study.Further more,The Trichinella spiralis larval distribution and number in the capsule,the sensitivity and the influenced factors of the Trichinella quarantine method which widespread used currently at domestic and international were also observed. Materials and Methods1.Trichinella and experimental animals Five species of Trichinella:Trichinella spiralis(T1),T.native(T2),T.britovi(T3),T.pseudospiralis(T4) and T.nelsoni(T7) were obtained from International Trichinella Referencre Centre(ITRC).Seven swine Trichinella isolates from Henan,Yunnan,Hubei,Xi'an,Harbin,Tongjiang and Tianjin.Male Kunming mice and Sprague Dawley(SD) rats were bought from the Experimental Animal Center of Henan Province.2.Acquire the genomic DNA from muscle larvae and PCR Genomic DNA was prepared according to a conventional method[phenyl-Chloroform method]from muscle larvae.A 419 bp region of the mitochondrial cytochrome c-oxidase subunitⅠ(COXⅠ) gene was amplied by PCR with a pair of primers[L6625(5'-TTYTGRTTYTTYGGNCAYCC-3'); H7005(5'-ACNACRTARTANGTRTCRTG-3')].3.PCR-RFLP After amplication,PCR products were puried.All of the amplicons were digested using restriction endonuclease Trull(Msel) according to the manufacturer's instructions.The digested products were electrophoresed in 3%agarose gel and visualised under BIO Imaging System.4.PCR-SSCP Before SSCP,all of the PCR products were puried.The 8% nondenaturing polyacrylamide gel was used to detect the products of SSCP.Silver staining method was used to visualize DNA fragments after electrophoresis.Obtain the allele frequency,genotype frequency,and polymorphism information content(PIC) by calculate.5.Trichinella spiralis larval distribution in muscles of rats and mice and its number in capsule Thirty male Kunming mice and thirty male SD rats were randomly divided into 3 groups(10 per group):light,moderate and heavy infection groups.Each group was orally inoculated with 1,5 or 20 muscle larvae of T.spiralis,respectively.All infected animals were slaughtered at 40 days post-infection.The muscles of lingual muscle,masseter muscle, intercostal muscle,diaphragmatic muscle,abdominal muscle,front legs muscle and hind legs muscle were compressed between two slides to be examined under microscope after weighted, to observe the larval burden(larvae per gram,lpg) and the number in capsule(nurse cell).6.The quarantine for the lowest T.spiralis lpg in meat Add T.spiralis larvae into pork(with out Trichinella) with 1～5 muscle lpg,test under microscope after artificial digestion method,to contrast the detection rates among the five groups.7.Pre-encapsulated larva(PEL) quarantine Three groups of mice were inoculated with 20,10 or 5 muscle larvae respectively,all mice were slaughtered at 18 days postinoculation, the artificial digestion method which recommended by lternation Commission Trichinellosis(ICT-digestion method),government standard 'diagnostic techniques for trichinella spiralis in swine(GB/T 18642-2002)'(GB-digestion method) and Baermarn's technique were used to quarantine the PEL,to compare the effectiveness of three methods.Results1.PCR The results of electrophoresis of PCR products of the Trichinella COXⅠgene showed that the band(419bp) of International Reference strains of five Trichinella species(T1,T2,T3,T4 and T7) was the same as seven Trichinella isolates from China.2.PCR-RFLP Electrophoresis of COXⅠgene PCR products digested by restriction endonuclease Trull(MseD showed that T2,T3,T4,T7 and Tianjin isolate have their own restriction fragments:T2(22bp,70bp,327bp),T3(92bp,327bp),T4(419bp),T7(62bp,64bp, 70bp,223bp) and Tianjin isolate(92bp,126bp,201bp).The other six isolates and T1 have the same restriction fragments(22bp,70bp,126bp,201bp).3.PCR-SSCP Three genotypes(AA,AB and BB) among different Trichinella isolates were observed by SSCP.Poland isolate was AA,Tianjin was BB,other isolates were AB.The frequency of AB genotype was the highest(0.75),the frequency of AA and BB genotypes were 0.125,and the frequency of A allele was equal to B(0.5).The PIC of different Trichinella isolates was 0.375.4.T.spiralis larval distribution in muscles of rats and mice and its number in capsule In rats with light infection,the larval burden in masseter muscle was the highest;In rats with moderate and heavy infection,the muscles contained the highest larval burden were diaphragmatic and lingual muscle,respectively.In mice with light infection,the larval burden in masseter muscle was also the highest;in both groups of mice with moderate and heavy infection,the larval burden in diaphragms was the highest.The frequency of capsules contained 1,2 and 3 larvae among 8028 capsules in infected rat muscles was 97.91%,1.95% and 0.14%,respectively.Out of 7559 capsules in infected mouse muscles,the capsules contained 1,2 and 3 larvae accounted for 97.33%,2.54%and 0.13%,respectively.The detection rate of capsules contained multiple(2～3) larvae in infected mouse muscles was obviously higher than that in infected rat muscles(χ²=5.644,P＜0.05).The detection rate of capsules contained multiple larvae in rats and mice has the elevating trend with the increase of infective dose(χ_rat²=42.656,P＜0.05;χ_mouse²=45.713,P＜0.05).The muscles of rats and mice with heavy infection accounted for 81.82(9/11)与100%(10/10)of the muscles contained the capsules with 3 larvae.The capsules contained more than 4 larvae(4 or more) were not detected.5.The result of the quarantine for the lowest T.spiralis lpg in meat For each 10 pieces of pork,when the lpg were 5,4 and 3,the detection rates of the ICT-digestion method were 100%;The rates were only 30%and 10%for the lpg 2 and 1.6.The effects of three methods for PEL quarantine Three groups of mice were slaughtered at 18 days post-infection,for the 15 pieces of meat in the group which infected with 20 muscle larvae,the detection rates of ICT-digestion method,GB-digestion method and Baermarn's technique were all 100%(χ₂₀²=0.000,P＞0.05).For the 15 pieces of meat in the group which infected with 10 muscle larvae,the detection rates of three methods were 93.33%,93.33%and 100%in order(χ₁₀²=1.645,P＞0.05);For the 30 pieces of meat in the group which infected with 5 muscle larvae,the detection rates of three methods were 63.33%, 90%and 100%(χ₅²=18.866,P＜0.05)。Conclusion1.The polymorphism of the COXⅠgene of seven swine Trichinella isolates from China and Poland isolate is moderate.These isolates in China which from Henan,Hubei,Yunnan, Xi'an,Harbin,Tongjiang are classified as one group,the Tianjin isolate is classified as another group.2.While the epidemiological investigation on murine Trichinella infection was carried out,masseter muscle is preferred for pathogen examination,the next is diaphragm or masseter muscle;The detection of the capsules contained 3 larvae during muscle biopsy or meat quarantine suggest the presence of heavy Trichinella infection. 3.The ICT-digestion method has a large omission factor at low-grade infection.For the PEL quarantine,the Baermarn's technique is obviously better than ICT-digestion method and GB-digestion method.