Lung flukes, paragonimus heterotremus and paragonimus westermani, in vietnam: morphology, genetics, biology and immunology diagnosis

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”

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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 2 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 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 3 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. 4 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 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 6 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; 7 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. 8 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 9 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 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 4 0 C 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 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 11 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 Sites Crab 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 Tri Da Krong Da Krong V. aluoiense 50 39 (78,0) 1-78 (10,1) Ta Long V. aluoiense 50 48 (96,0) 12-608 (80,0) Huong Hoa Huong Son V. aluoiense 50 50 (100,0) 8-500 (140,8) Tan Thanh V. 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, 12 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 Site Crab species Rate (%) and intensity of infection (metacercaria / crab) of species P. vietnamensis P. heterotremus P. bangkokensis P. westermani P. proliferus Luong Son I. tannanti 8,6 (1-4) 82,8 (4-504) An Lac I. tannanti 6,0 (1-2) 69,2 (1-360) 1,5 (2-2) 13,8 (1-33) Da Krong V. aluoiense 4,0 (1-2) 6,0 (1-2) 78,0 (1-78) Ta Long V. aluoiense 8,0 (1-4) 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) D. haii 12,0 (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. 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, 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). 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 sn
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