Summary of Engineering Doctoral thesis: Lung flukes, paragonimus heterotremus and paragonimus westermani, in Vietnam: morphology, genetics, biology and immunology diagnosis

The objective of the thesis is to know about two species of SLP P.heterotremus and P. westermani, providing a scientific basis for the diagnosis and prevention of SLP disease, contributing to protecting public health.

MINISTRY OF EDUCATION VIETNAM ACADEMY OF SCIENCE AND TRAINING AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

LUU ANH TU

LUNG FLUKES, PARAGONIMUS HETEROTREMUS AND

PARAGONIMUS WESTERMANI, IN VIETNAM:

MORPHOLOGY, GENETICS, BIOLOGY AND

The thesis is completed at: Graduate University of Science and

Technology - Vietnam Academy of Science and Technology

Supervisors: 1 TS Pham Ngoc Doanh

2 TS Bui Khanh Linh

of Science and Technology

Time: Date… month … 2018

This thesis can be found at:

- The library of Graduate University of Science and Technology;

- National Library of Vietnam

INTRODUCTION

1 The necessity of the research

Lung flukes of the genus Paragonimus cause paragonimiasis

which affect human and animal health The main reason of infection

is due to ingestion of metacercariae from second intermediate host or juvenile flukes from paratenic host Symptoms of lung fluke are more likely to be misdiagnosed with pulmonary tuberculosis or other pulmonary disease, producing difficulties to diagnose and treat the disease (Blair et al., 1999)

In Vietnam, Paragonimus and paragonimiasis have been studied

for more than 20 years (Vien et al., 1994; De et al., 1998-2003;

Doanh et al., 2005-2013) To date, 7 Paragonimus species have been reported (Doanh et al., 2013) Of which, P heterotremus is prevalent

in the north provinces and P westermani is prevalent in the north

central provinces Both species can infect humans However, many issues of two species have not been unknown Therefore, we

conducted a study on “Lung flukes, Paragonimus heterotremus and

Paragonimus westermani, in Vietnam: morphology, molecular

biology and immunological diagnosis”

2 Objectives

The main purpose of this topic is to increase the knowledge of

two species, P heterotremus and P westermani, in order to provide

a scientific basis for diagnosis and prevention of paragonimiasis, contribute to public health protection

The specific objects:

1 To determine the best method for detection of Paragonimus

metacercariae in crabs and to determine the status of

Paragonimus metacercariae infection in crabs in Lao Cai, Yen

Bai and Quang Tri provinces

2 To determine the morphological diversity of metacercariae and

molecular diversity of P heterotremus and P westermani

3 To identify the biological characteristics of P heterotremus and

P westermani

4 To set up dot-ELISA to rapidly diagnose paragonimiasis in the field

3 Research contents

3.1 Determination of the best method of crab examination for

Paragonimus metacercariae and investigation of metacercarial

infection in crabs in Lao Cai, Yen Bai, Quang Tri

3.2 Study of the morphological diversity of metacercariae and

molecular variation of P heterotremus and P westermani

3.3 Identification of the biological characteristics of two species

- Identification of the first intermediate host and definitive hosts

- Development of lung fluke in the definitive and paratenic hosts

- Observation of the vitality of metacercariae

3.4 Set up dot-ELISA to diagnose paragonimiasis

- Determination of the specificity and sensitivity of dot-ELISA

- Determination of antigen concentration of the reaction

- Determination of the reaction time at different temperature conditions

4 Scientific and practical meaning of the topic

4.1 Scientific meaning: This study provides the scientific

information on the morphology of metacercariae, genetics and

biological characteristics of P heterotremus and P westermani in

Vietnam

4.2 Practical meaning: This study provide the scientific basis for

the prevention of Paragonimus infection and established dot-ELISA

for rapid diagnosis of paragonimiasis in the field, contributing to public health protection

5 New contributions of the topic

5.1 Detected metacercariae of P heterotremus in the Central and

P westermani in the North, and described the morpholigical

diversity of metacercariae of the two speices

5.2 Analysed the genetic diversity of P heterotremus and

P westermani

5.3 Identified the first intermediate hosts and cercaria larvae of two

species P heterotremus and P westermani, and also

P proliferus

5.4 Identified wild cats as the definitive host of Paragonimus spp in

DaKrong district

5.5 Identified that domestic dog were not infected with

P westermani and domestic cat were infected with

P westermani with susceptibility lower than that of

P heterotremus

5.6 Identified mice as paratenic host in life cycle of P heterotremus

and P westermani in Vietnam

5.7 The vitality of metacercariae depends not only on the culture

medium and temperature, but also on the density of

metacercariae

5.8 Established dot-ELISA and determined the reaction time in

different temperature conditions, which can be applied to rapidly

diagnose paragonimiasis in the field

6 Thesis structure

The dissertation composed of 111 pages, including introduction 3

pages, 21 pages of literature review, 15 pages of materials, content and

methodology, 54 pages of results and discussion, 3 pages of conclusions

and suggestions, 2 pages of new contribution and publications, 13 page of

references The thesis has 21 tables, 54 images and charts, 132 references

CHAPTER 1 OVERVIEW 1.1 Lung flukes of the genus Paragonimus

To date, more than 50 lung fluke species of the genus

Paragonimus have been described The life cycle of lung fluke

requires three to four hosts The definitive hosts are wild and

domestic animals, especially cats and dogs, and also human being

The first intermediate hosts are freshwater snails, and the second

intermediate hosts are crab and some freshwater shrimp species

(Blair et al., 1999) Paratenic hosts are some crab eaters

Diagnostic methods for detection of paragonimiasis include four

groups: parasitology, serological/immunological, imaging and

molecular methods

1.2 Paragonimus heterotremus and Paragonimus westermani

1.2.1 Paragonimus heterotremus

P heterotremus is distributed in South Asia, Southeast Asia,

and Southern China, and causes human disease in these areas The

first intermediate hosts are member of the family Assimineidae and

Pomatiopsidae The second intermediate hosts are six crab species of

the family Potamidae The natural definitive hosts are cats and

squirrels in Thailand, experimental animals are dogs, cats, mice and

rabbits Mice act as paratenic hosts (Blair et al 1999)

1.2.2 Paragonimus westermani

P westermani is widely distributed in Asia, with a great diversity

of morphology, genetics, and biology Paragonimus westermani has

two types of triploid (3n) and diploid (2n) Life cycle requires 3-4

host species The first intermediate hosts are species of Brotia and

Semisulcospirus The second intermediate hosts are 8 species of

shrimp and 40 species of crabs The definitive hosts are many species

of mammals The susceptibility to P westermani varies markedly

among geographic populations Remarkably, people infected with

P westermani are limited to East Asia and the Philippines The

paratenic hosts include pigs, wild pigs and deer (Blair et al., 1999;

Yoshida et al., 2016)

1.3 Research on Paragonimus and paragonimiasis in Vietnam

Paragonimiasis in Vietnam has been studied since 1994 So far,

seven species have been found in the northern and central provinces

Of these, three species (P heterotemus, P westermani and

P skrjabini) have the potential to infect humans Paragonimus

skrjabini species was found in Thanh Hoa province with low

infection rate; P heterotremus is prevalent in the North;

P westermani species is prevalent in the Central (Doanh et al.,

2013) Many problems of P heterotremus and P westermani have

not been elucidated (Doanh et al., 2013) In addition, new cases of paragonimiasis are still being discovered Therefore, rapid and simple diagnostic technique in the field is necessary to cure diseases

in a timely manner, contributing to the protection of public health These issues will be addressed in this thesis

CHAPTER 2 MATERIALS AND METHODS 2.1 Materials and study locations

2.1.1 Materials

P heterotremus and P westermani

2.1.2 Study locations

Luong Son commune (Bao Yen district, Lao Cai province) and

An Lac commune (Luc Yen district, Yen Bai province) where the

prevalence of P heterotremus metacercariae is high and four

communes of Quang Tri province are Huong Son and Tan Thanh (Huong Hoa district), and Da Krong and Ta Long communes (Da

Krong district) - where P westermani infection in second

intermediate host is very high

2.1.3 Study time: from 10/2014 to 10/2017

2.2 Approach and experimental design

Detection of metacercariae in second intermediate hosts is fastest and is the most accurate indicator of the distribution of

Paragonimus Therefore, at the study sites, crabs will be examined

first to identify the sites with the highest prevalence of metacercariae

of two species P heterotremus and P westermani for further

identification of the natural definitive and first intermediate hosts Combination of morphological and molecular methods to identify

both Paragonimus and their hosts Metacercaria were collected for

other morphological, genetic and biological studies

2.3 Methods

2.3.1 Methods of collecting metacercariae

Determination of sedimental time of refining method: Take a crab caught from non-endemic area Remove the crab shells, pestle

in a mortar

Adding 50 P westermani metacercariae in freshly grounded

crab and 250 ml of water, stir well and filter through a 1 x 1 mm sieve into a 300 ml plastic cup Stand for the test time, then pour ½ portion of the solution to another cup Adding water to the residue Repeat procedure of filtering-sediment until the sediment is clear and can be seen under a microscope The tests will be done with sedimentation time between filters of 3 minutes, 2 minutes and 1 minute by using the stopwatch Experiments were repeated 3 times Examination of the last sediment under a microscope to find the metacercariae

The most appropriate sedimentation time is the shortest time to recover 50 metacercariae in the sediment of the final filter Do the

same procedure with the metacercariae of P heterotremus

Comparison of two methods of filtering-sediment and pressing between two glasses regarding the time and the number of metacercariae obtained

2.3.2 Investigation of infection rates of metacercariae

Catching crabs at streams, at least 50 crabs for each sites Identification of crabs is according to Dang Ngoc Thanh and Ho Thanh Hai, 2012

Using the best method determined to examine the crabs for

the rate and intensity of infection

2.3.3 Morphological study of metacercariae: Based on shape, size

and shell thickness Metacercariae was morphologically identified according to Doanh et al., 2013

2.3.4 Molecular variation of lung fluke

Gene selection: for P heterotremus, ITS2 and mitochondrial CO1 gene were selected, for P westermani ITS2 and 16S

mitochondrial gene were selected, as this gene can discriminate

between 2n/3n types of P westermani

Molecular analyses include following steps: Total DNA extraction; PCR reaction to replicate target sequence with selective primer; purification of PCR product; direct sequencing; comparison

of obtained sequences with available sequences in GenBank by BLAST and phylogenetic tree were generated by MEGA6 software

2.3.5 Infections to the hosts

Two species were experimentally infected for 10 white mice with 50 metacercariae/mouse After 1 to 2 months, the mice was sacrified for juveline worms and transfer to cats

P westermani were infected to 4 dogs and 8 domestic cats with

30-50 metacercariae/host Monitoring of laboratory animals, periodic examination of faeces to find eggs by the sedimentaion method, determine the time of development of flukes in animals

2.3.6 The morphology study of mature fluke: Staining the specimen

by carmine alumine staining method Measure the size of flukes and organs (suckers, testicles and ovaries)

2.3.7 Identification of the natural host of the lung fluke

- Identification of first intermediate hosts: snail species were collected at the sites with high infection rate in crab host, and were identified according to Dang Ngoc Thanh et al., 2006 Sheding and squeezing between two glasses were done to collect cercariae The cercariae were identified by analyzing ITS2 sequences The snail infected with Paragonimus larvae were re-identified by CO1 sequences

- Identification of the definitive host: Examination of stool

samples of domestic dogs and wild animals for Paragonimus eggs

Eggs will be identified by ITS2 sequences Stool samples containing

Paragonimus eggs were used to identify the host by D-Loop region

of the mitochondrial genome

2.3.9 Set up a dot-ELISA assay

- Antigen of P heterotremus and P westermani

- 30 serum samples of paragonimiasis patients infection, 30 samples of patients infected with fascioliasis, clonorchiasis, pulmonary tuberculosis and normal human serum were used to check for cross-reactivity

- Dot-ELISA technique were performed according to Ito and Sato

1990 Reactions at different concentrations of antigen, temperature, and time to provide optimum conditions

2.4 Statistic analysis: Excell, SPSS

CHAPTER 3 RESULT AND DISCUSSION 3.1 Comparison of two methods of crab examination and infection rates of metacercariae in crabs

3.1.1 Comparison of two methods of crab examination

3.1.1.1 Determination of sedimentation time of refining method

With crab-filtering method, the time for sedimentation to the

bottom of P westermani is 2 minutes, P heterotremus is 3 minutes Metacercariae of P heterotremus is smallest compared to other lung

fluke species (Doanh et al., 2015), so 3 minute is the most appropriate time

3.1.1.2 Comparison of two methods

The time to examine a crab by filtering method was 33-38 minutes (mean 35,5 minutes) which was faster than that of pressing

crab between two glasses (76-85, mean 80.3 minutes) (P <0.005)

Of the 10 crabs collected in Yen Bai, 902 metacercariae was collected by filtering method, while pressing between two glasses

collected 252 metacercariae (P <0.001)

3.1.2 Infection rate and intensity of metacercaria in crabs

At the study sites, three species of crabs were found:

Indochinamon tannanti in Yen Bai and Lao Cai, and two species of Vietopotamon aluoiense and Donopotamon haii in Quang Tri

province

All 3 crab species were infected with Paragonimus

metacercariae The incidence and intensity of infection is shown in Table 3.4

Table 3.4 Prevalence of metacercariae in crabs

species

Number Number

of infection (%)

Intensity Province District Commune

Lao Cai Bao

Yen

Luong Son I tannanti 70 58 (82,8) 4-504

(63,8) Yen Bai Luc

Yen

An Lac I tannanti 130 91 (70,0) 1-362

(19,7) Quang

aluoiense

50 5 (10,0) 1-6 (3,4)

D haii 50 6 (12,0) 1-3 (2,2)

In Quang Tri, metacercariae of four species (P westermani,

P bangkokensis, P proliferus and P heterotremus) were found, of

which P westermani infection was highest In two communes (An Lac and Luong Son) metacercariae of 4 species (P heterotremus,

P vietnamensis, P westermani and P bangkokensis) were found, of

which the infection rate of P heterotremus was highest (table 3.5)

Table 3.5 Infection rates of Paragonimus species in crabs

82,8 (4-504)

69,2 (1-360)

1,5 (2-2)

13,8 (1-33)

6,0 (1-2)

78,0 (1-78)

96,0 (12-608) Huong

Son

V

aluoiense

4,0 (1-3)

100,0 7-500

4,0 (1) Tan

Thanh

V

aluoiense

2,0 (1)

10,0 (1-5)

(1-3)

Note: The number in parentheses is the range of intensity

3.2 Metacercarial morphology and molecular genetics

3.2.1 Paragonimus westermani

Metacercariae of P westermani collected from Yen Bai were

relatively uniform in morphology while those collected from Quang

Tri differed in their size and shape (P <0.001), and were divided into

five forms (Figure 3.5a-e)

Figure 3.5 P westermani metacercaria collected from Quang Tri

ITS2 sequences of P westermani differed 1-2 (0.2-0.4%)

nucleotide positions, forming one group (Figure 3.6) The 16S

sequences of P westermani collected from Quang Tri and Yen Bai

provinces varied by 1.1-1.4%, forming two groups in the same clade

(Figure 3.7) The P westermani of Vietnam belong to 2n type

3.2.2 Paragonimus heterotremus

Metacercariae of P heterotremus collected from Yen Bai and Lao

Cai have a diversity of sizes (Figure 3.8) In Quang Tri, 2 metacercariae were obtained with the oval shape, size 226-252 x 218-236 μm

Figure 3.8 Metacercaria of P

heterotremus collected from Yen Bai

and Lao Cai

Molecular analyses revealed that ITS2 and CO1 sequences of

two samples of P heterotremus from Quang Tri were completely

identical to each other and highly similar to samples from the North (Figures 3.10 and 3.11)