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.
1 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 IMMUNOLOGY DIAGNOSIS Major: Parasitology Code: 62.42.01.05 SUMMARY OF DOCTORAL THESIS Hanoi, 2018 The thesis is completed at: Graduate University of Science and Technology - Vietnam Academy of Science and Technology Supervisors: TS Pham Ngoc Doanh TS Bui Khanh Linh Reviewer 1: Reviewer 2: Reviewer 3: The doctoral thesis will be defended at the Evaluation Committee of Graduate University of Science and Technology, Vietnam Academy 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 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, 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” 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: 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 To determine the morphological diversity of metacercariae and molecular diversity of P heterotremus and P westermani To identify the biological characteristics of P heterotremus and P westermani To set up dot-ELISA to rapidly diagnose paragonimiasis in the field 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 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 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 Thesis structure The dissertation composed of 111 pages, including introduction pages, 21 pages of literature review, 15 pages of materials, content and methodology, 54 pages of results and discussion, pages of conclusions and suggestions, pages of new contribution and publications, 13 page of references The thesis has 21 tables, 54 images and charts, 132 references CHAPTER 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 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 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 x 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 minutes, minutes and minute by using the stopwatch Experiments were repeated 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 to months, the mice was sacrified for juveline worms and transfer to cats P westermani were infected to dogs and 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 10 Paragonimus eggs were used to identify the host by D-Loop region of the mitochondrial genome 2.3.8 Vitality of metacercariae - Metacercariae of each species was cultured in physiological solution, at different temperature conditions: room temperature and 40C at densities of 5, 50, 100, 200 metacercaria/1ml Periodically observe under a microscope to determine the excystation and viability of the metacercariae 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 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 minutes, P heterotremus is minutes Metacercariae of P heterotremus is smallest compared to other lung fluke species (Doanh et al., 2015), so minute is the most appropriate time 3.1.1.2 Comparison of two methods 13 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, 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) 14 Figure 3.6 Molecular evolution relationships of P westermani populations based on ITS2 sequences were constructed using the Maximum Likelihood method Figure 3.7 The molecular evolution relationships of P westermani populations based on the 16S gene sequences were constructed using the Maximum Likelihood method 15 Figure 3.10 The molecular evolution relationship of P heterotremus populations are based on the ITS2 sequence Figure 3.11 The molecular evolution relationship of P heterotremus populations are based on the CO1 sequence 16 3.3 Biological characteristic of Paragonimus heterotremus and Paragonimus westermani 3.3.1 First intermediate hosts Cercaria belonging to the microcercaria group were found in small snails (4 mm) of subfamily Triculinae in Lao Cai, Yen Bai and Huong Son (Quang Tri), and large snails (4 cm) like Sulcospira in Quang Tri CO1 sequence analysis of the large snail specimens in Quang Tri province identified them as Sulcospira quangtriensis CO1 sequences of small snails had the highest similarity (90-91%) with Gammatricula fujiansis (AF213342) Small snails in Yen Bai and Lao Cai were similar to each other, but were different from the snail in Quang Tri at 12.6% The distance between species of Gammatricula varied from 6.4% to 12.5% Therefore, it can be confirmed that small snails from Quang Tri are different from snails collected from Yen Bai and Lao Cai On the phylogenetic tree, they formed different branches closed to genus Gammatricula and Tricula of subfamily Triculinae with low bootrap values Therefore, these specimens should be placed in the Gammatricula genus or other of the subfamily Triculinae At present, we temporarily named the snails species from Lao Cai and Yen Bai as Triculinae gen sp.1 and from Quang Tri as Triculinae gen sp.2 The ITS2 of larvae collected from Triculinae gen sp1 from Lao Cai and Yen Bai was completely identical (100%) to P heterotremus, and that from snail Triculinae gen sp.2 was P proliferus, and that from snail S quangtriensis was P westermani Morphology of larvae in intermediate host Cercaria of species is very similar in shape and size (P> 0.05) Rediae of three species is similar in size ((P > 0.05) However, they can be distinguished by the intestinal length and the number of cercariae in each redia The redia of P westermani has a long intestine equal to 70% of the body, the other two species have a short 17 intestine equal to 20% of the body length ((P