Trichinella spp and clonorchis sinensis

Liu Mingyuan

OIE Collaborating Centre for Food-Borne Parasite from the Asian-Pacific Region

Institute of Zoonosis, Jilin University, China

Trichinella spp

Infection and infestation of human and animals

Life cycle of Trichinella

In the world

• > 150 animals species can be infected• > 10030 human cases, estimated more than 10 million people

infected. ICT reported 1995-1997.

In the world

• > 0.570 billion Euro in EU, 1 billion US＄ in USA, 20 billion US＄ in China for pig inspection

Pork product in world

China

European UnionUSA

Russia

Mexico

South Africaothers

0.73 billion pig slaughter

Trichinellosis in China

• > 14 mammal animal species infected.• > 27 thousand human cases since 1964, 40 millions

people with sera-positive (2005, national surveillance).• > 0.64 billion pigs for Trichinella inspection per year.

20 millions 10 years ago, 1995

94,3 %

5,7 %

Reservoir for human cases in China

Recent outbreaks in Yunnan province, China, 2009

Trichinella species, evolution and migration in China

Identified by the PCR and cross breeding

ISS531, ISS532, ISS533, ISS534, ISS535, ISS536 。

9 micro-setalite markers and 1/5 mitochondrion genome. More than 400 isolates in the world cooperated with USDA, Dr. Benjamin

Kimura evolution tree for T. spiralis

migration

Antigen genes screening in different

development stages of

T. spiralis

Trichinoscopy: 3-5 /g

Digestion: 3/g

Serological methods: 0.01/g

What’s the

problem

More than 1.4 billion pigs were slaughteredin world each year.

０ 3 9 12 15 18 21－ 35 (days)

ES antigen

Life cycle

Purpose of the study

Pigs antibody detection

ES antigen

Life cycle

Purpose of the study

Pigs antibody detection

Blind area

０ 3 9 12 15 18 21－ 35 (days)

ES antigen

Life cycle

Purpose of the study

Pigs antibody detection

Blind area

０ 3 9 12 15 18 21－ 35 (days)

Pig anti-trichinella serum

Positive genes (clones) from 6h old ML in intestine cDNA library

Code

Number

Clones

Member

Homology analysis of deduced protein

T626-55 20 Cystatin like protein （Trichinella spiralis) ［ABY60755.1］, 100%

T626- 1 2 Putative porin precursor （Trichinella spiralis) ［ABY60749.1］, 100%

T626-19 1 Putative serine protease （Trichinella spiralis) ［ABY60762.1］, 100%

T626- 7 1 No significant similarity

T626-16 1 BAC clone TS195-31H12 from chromosome DNA （Trichinella spiralis)

［AC193814.3］, 96%

T626-20 1 mitochondrion DNA （Trichinella spiralis) ［AF293969.1］, 98%

T626-26 1 Hypothetical protein Bm1_10535 （Brugia malayi) ［EDP37588.1］, 56%

T626-29 1 Rieske iron-sulfur protein 1 （Graphocephala atropunctata) ［ABD98746.1］,

70%

T626-30 1 Temporarily assigned gene name protein 59 （Caenorhabditis elegans)

［AAB52260.3］, 60%

T626-32 1 Hypothetical protein TRIADDRAFT_53524 （Trichoplax adhaerens)

［EDV28185.1］, 53%

T626-33 1 Ubc protein （Rattus norvegicus) ［AAI03478.1］, 92%

T626-42 1 GM13181 protein （Drosophila sechellia) ［EDW44127.1］, 95%

T626-48 1 Putative ATP binding protein （uncultured marine bacterium Ant4E12)

［ABC25270.1］, 48%

From Ad3 cDNA library

Clone name (Identical clones)

Accession no.Size(bp/aa)

Sequence similarity Accession no.BLASTXE value

Ad5 (2) DQ864973 1307/373 T. spiralis serine proteinase inhibitor AAF63473 0.0

WN10(23) EU263325 1352/406 T. spiralis hypothetical ORF 9.10 AAB48491 2e-135

Ad147 (4) EU331362 950/237 T. spiralis hypothetical ORF 9.10 AAB48491 8e-12

Ad53(1) EU263318 1453/451 T. spiralis 53 kDa excretory/secretory antigen AAA97512 7e-11

Ad67(1) EU263319 1095/312 Trichuris trichiura putative porin precursor AAC04763 2e-11

Ts Adsp-1(1) EU263326 589/193

Trypsin-like serine protease family AAK16516

2e-24

Ts Adsp-2(3) EU263327 1594/429 3e-44

Ts Adsp-3(1) EU263328 1375/429 3e-46

Ts Adsp-4(1) EU263329 1539/429 5e-46

Ts Adsp-5(5)（L23)

EU263330 1534/429 2e-46

Ts Adsp-6(1) EU263331 1205/370 4e-35

Ts Adsp-7(3) EU263332 1372/429 3e-44

Ts Adsp-8(1) EU263333 1399/429 3e-44

Ad42(1) EU263322 907/296 Caenorhabditis elegans paramyosin NP_492085 1e-69

Ad71(1) EU331363 751/243 Plasmodium yoelii Ran-binding protein EAA19671 3e-07

Ad76(1) EU263309 886/209 Caenorhabditis elegans calcium-sensing receptor NP_501400 1e-21

Ad62(1) EU263308 1165/388 No sequence similarity - -

Ad2(1) EU263321 735/174 No sequence similarity - -

From NBL cDNA library

Clone name

(identical

clones)

Accession

no.Size

(bp/aa)Sequence similarity Accession no.

BLASTX

E value

NBL84 (2) EF041139 1379/459 T. spiralis glutamic acid rich

protein cNBL 1500

T. spiralis glutamic acid rich

protein cNBL 1700

AAM19759

AAM19760

0.0

0.0

NBL122 (2) DQ864973 1307/373 T. spiralis serine proteinase

inhibitor

AAF63473 0.0

NBL39 (2) EU263320 474/117 T. spiralis hypothetical ORF

11.30 precursor

AAB48488 3e-45

NBLWN18 (70) - 1587/465 T. spiralis newborn

larvae-specific

serine protease SS2

AAK16520 1e-58

NBL50 (2) EU263319 1095/312 Trichuris trichiura putative

porin precursor

AAC04763 2e-11

NBL43(1) EU263324 1276/425 Homo sapiens heat shock

protein HSP 90-alpha 2

CAI64495 e-166

NBLWN5(1) EU263316 1552/475 Plasmodium yoelii

glutamine-asparagine rich

protein

EAA18721 9e-13

NBLWN11(1) EU263317 1540/389 Caenorhabditis elegans

putative RNA binding G-

patch domain protein

NP_497893 7e-24

From ML cDNA library

Clone name

(identical

clones) Accession

no.

Size

(bp/aa)Sequence similarity

Accession

no.

BLAST

X

E value

MLWM5 (10) DQ864973 1307/373 T. spiralis serine proteinase

inhibitor

AAF63473 0.0

ML42(2) EU263312 678/170 T. spiralis hypothetical ORF 17.20

T. pseudospiralis 21 kDa ES protein

AAB48489

AAF79206

3e-85

2e-63

ML3(1) EU263323 671/158 Ostertagia ostertagi globin-like ES

protein F6

CAD20463 2e-07

ML59(1) EU263313 447/113 Arabidopsis thaliana 60s acidic

ribosomal protein P1

BAB11203 9e-12

ML41(1) EU263311 563/153 Dunaliella tertiolecta nucleoside

diphosphate kinase

AAK38732 2e-56

ML63(1) EU263314 1170/192 Homo sapiens putative G-protein coupled

receptor SH120

CAI13224 9e-43

ML1(1) EU263310 1311/437 Mus musculus nuclear matrix protein

SNEV

NP_598890 e-135

ML64(1) EU263315 767/199 No sequence similarity - -

T314

T660T42E10

T668

Prokaryotic expression

Western blot: fusion proteins with 26 dpi serum

Western blot with 60 dpi serum

T626-55

L23

WN18

WM5

ELISA

Strips

Enlarged nucleiInvasion

cell

T626-55

Enlarged nuclei

Invasion cell

WM 5

Enlargednuclei Invasion

cell

ZH68

EU patent

T.spiralis-N10-AAO17050 TSNBL.DNaseII-8.AY790261

TSNBL.DNaseII-3.AY790256 TSNBL.DNaseII-9.AY790262 TSNBL.DNaseII-5.AY790258 T.spiralis-NBLSS1-AAK16519

TSNBL.DNaseII-4.AY790257 TSNBL.DNaseII-1.AY790254 TSNBL.DNaseII-2.AY790255

TSNBL.DNaseII-6.AY790259 TSNBL.DNaseII-7.AY790260

TSNBL.DNaseII-10.AY790263 TSNBL.DNaseII-11.AY790264

P43-ISS534 TSAd.DNaseII-10bankit690445 TSAd.DNaseII-8bankit703732 TSAd.DNaseII-9bankit690443

TSAd.DNaseII-7bankit703730 TSAd.DNaseII-5bankit690441 TSAd.DNaseII-3bankit703724 TSAd.DNaseII-4bankit690437 TSAd.DNaseII-2bankit690427 TS.Ad-AAK85403 TSAd.DNaseII-1bankit703712

0.1

Finding of DNase II families in T. spiralis

T3223-6 from adultImmunohistochemistry

T3223-6 from adult

Cord cell

T3223-6 gene

Macrophages launch DNA from genome or mitochondrion DNA to catch and kill bacteria. Streptococcus can secrete DNase to digest

the DNA for escaping

A B

C D

How Trichinella escape the capture of Macrophages

How Trichinella escape the capture of Macrophages

A

C

B

D

Incubated with Anti-Trichinella serum

How be captured and killedHow the snake eat elephant

How be captured and killedHow the snake eat elephant

Existence is reasonable

G. Hegel, famous philosopher

Colitis

Diabetes

Rheumatoid arthritis

Pollen

Asthma

Obesity

Immunomodulator

（一）立项依据

INFγ

Quick copy pathogensbacteria、virusprotozoa、fungi

IL4, IL13

Mutiple cell pathogensHelminth, tumor

Th1

Pre-inflammationIL6, IL12, TNF,

INFγ, IL1 , ROI

Th2

Anti-inflammationIL10, TGFβ, IL4, IL5,

IL13

47

protozoa

（一）立项依据

Th1

促炎反应IL6, IL12, TNF,

INFγ, IL1 , ROI

Th2

抑炎反应IL10, TGFβ,

IL4, IL5, IL13

炎症性肠病（IBD）Ⅰ型糖尿病多发性硬化症

48

INFγ IL4, IL13

Quick copy pathogensbacteria、virusprotozoa、fungi

Mutiple cell pathogensHelminth, tumor

Hygiene hypothesis

Not too dirty not too clean

Helminth may be a kind of pet with human forever

Clonorchis sinensis

Life cycle of Clonorchis sinensis

Adult worm

Egg

Metacercariae

Clonorchis sinensis

Liver damage

International Arctic Research Center

First classes of bio-pathogens inducing cancer

6 fishponds were investigated

Fish number Metacercariae /gFishpondsPositive percentage

Metacercariae in fish meat

Digested by pepsin

cDNA liboratories of Clonorchis sinensis

Immunoscreening by human positive serum

Positive clones selected

Number Code Protein Homology (%) Clones

9-9 glycine rich antigen 2 (Clonorchis sinensis) [AAM18463.1] 87% 11

16-26 proline rich antigen 2 (Clonorchis sinensis) [AAM18465.1] 55% 10

12-6 proline rich antigen 2 (Clonorchis sinensis) [AAM18465.1] 84% 2

12-4 antigen Cs44 (Clonorchis sinensis) [AAD33058.1] 98% 5

9-27 antigen Cs44 (Clonorchis sinensis) [AAD33058.1] 53% 3

16-4 predicted protein (Nematostella vectensis) [XP_001634591.1]

39% 7

9-16 transcription elongation factor GreA (Bartonella quintana str.

Toulouse) [YP_032532.1]32% 1

12-1 similar to NM_132090 CG3446 gene product in Drosophila melanogaster (Schistosoma japonicum) [AAP05955.1]

52% 1

16-28 predicted protein (Nematostella vectensis)[XP_001627036.1]

27% 1

16-3 PREDICTED: hypothetical protein (Pan troglodytes) [XP_001137791.1]

30% 1

J31 SJCHGC06649 protein (Schistosoma japonicum) [AAW25164.1]

60% 1

16-10 SJCHGC02535 protein (Schistosoma japonicum) [AAW25739.1]

43% 1

12-5 Unknown 1

New Conception

Trematode may be a sticker but not a sucker

Dr. Isabelle VALLEE

Contact point of CC in France

Dr. Alvin Gajadnar

Contact point of CC in Canada

Thanks for your attention

DNA RNA ProteinsTRANSCRIPTION TRANSLATION