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ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 2  |  Page : 176-178  

Evaluation of paragonimus metacercariae isolated from Maydelliathelphusa Lugubris and morphological characterization of Paragonimus Westermani recovered from an experimental model


Department of Microbiology, Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim, India

Date of Submission19-Mar-2020
Date of Decision16-Apr-2020
Date of Acceptance13-Jul-2020
Date of Web Publication22-Jul-2020

Correspondence Address:
Bhamani Devi Tongbram
Department of Microbiology, Sikkim Manipal Institute of Medical Sciences, Tadong, Gangtok - 737 102, Sikkim
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_61_20

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   Abstract 


Background: Humans acquire Paragonimus westermani (lung flukes) infestation by consumption of crabs and crayfish harboring Paragonimus metacercariae. This study was designed to evaluate the prevalence of P. westermani infection in fresh water crabs found in Manipur, India. The morphological characterization of adult fluke recovered from Wister rats infected with P. metacercariae was also studied. Materials and Methods: The freshwater crabs were collected from the mountain streams of Churachanpur district, Manipur, India. P. metacercariae were harvested from the crab extract under a stereomicroscope. To recover adult flukes, twenty P. westermani-type metacercariae were injected intraperitoneally to Wister rats. The adult fluke recovered from Wister rats were stained with borax carmine, and the whole mount was used for morphological assessment. Results: Crabs of species, Maydelliathelphusa lugubris, were found to harbor Paragonimus heterotremus- and westermani-type metacercariae. Two migratory flukes were recovered from the lung surface of the Wister rats injected intraperitoneally with metacercariae. The morphological features of the mounted specimen of adult fluke recovered showed six lobed ovaries with terminal branching and five lobed testes. On the basis of the morphological features, the species was identified as P. westermani despite the atypical features of its metacercariae. Conclusion: Here, we report the role of M. lugubris fresh water crab species as natural permissive hosts of lung fluke (P. westermani) in Manipur region of India.

Keywords: Fresh water crabs, lung fluke, Maydelliathelphusa lugubris, metacercariae, Paragonimus westermani, parasitic infestation


How to cite this article:
Tongbram BD, Singh TS, Tsering DC. Evaluation of paragonimus metacercariae isolated from Maydelliathelphusa Lugubris and morphological characterization of Paragonimus Westermani recovered from an experimental model. J Nat Sc Biol Med 2020;11:176-8

How to cite this URL:
Tongbram BD, Singh TS, Tsering DC. Evaluation of paragonimus metacercariae isolated from Maydelliathelphusa Lugubris and morphological characterization of Paragonimus Westermani recovered from an experimental model. J Nat Sc Biol Med [serial online] 2020 [cited 2020 Aug 12];11:176-8. Available from: http://www.jnsbm.org/text.asp?2020/11/2/176/290495




   Introduction Top


Lung flukes of the Paragonimus genus contribute to food-borne parasitic zoonosis worldwide and are endemic to several regions in Asia, Africa, America, and Russia. Nearly, fifty Paragonimus species are described, 15 of which are known to cause infections in humans.[1] The prevalence of Paragonimus westermani in many mammals is reported from several parts of mainland India.[2],[3],[4],[5] However, the detailed morphological description of the adult flukes and its pathobiology and the molecular characteristics of the Paragonimus species occurring in India is lacking. Different Paragonimus species such as Paragonimus hueitungensis, Paragonimus heterotremus, Paragonimus skrjabini,[6],[7],[8],[9] and Paragonimus macroorchis[10] were reported in the north-eastern region of India. P. heterotremus[11],[12] is reported as the most prevalent cause of human paragonimiasis in the north-eastern region of India. More recently, the occurrence of P. westermani is also reported in Arunachal Pradesh, Meghalaya, and Assam, India.[13],[14] However, its association with human pulmonary paragonimiasis was described in 2011 in a female patient from Moreh, Manipur, India.[15] Currently, at least two Paragonimus species, i.e., P. heterotremus and P. westermani, are known to cause human paragonimiasis in India. In the present study, we surveyed the Moreh and Churachandpur districts of Manipur, India, to examine the prevalence of P. westermani metacercariae in various fresh water crab species. Further, Wister rats were injected with the metacercariae to recover adult flukes for morphological characterization.


   Materials and Methods Top


Collection and examination of freshwater crabs for Paragonimus metacercariae

Study site

Churachandpur district (stretches between 93°15´E and 94°45´E longitudes and between 24°N and 24°30´N latitudes) is located in the southwestern region of Manipur. This district is mainly drained by the Barak, Khuga, Leimatak, and Tuivai rivers. Several crab species such as Potamiscus manipurensis and Maydelliathelphusa lugubris[16] are abundant in the mountain streams of this region. The crabs were collected from small tributaries of these rivers during July to October 2018 and brought back to the laboratory for study. The crabs were classified and grouped by species. The crabs in groups of 5–10 (depending on body size) were extracted using a mincer and then digested with artificial gastric juice. The extract was filtered through stainless steel filters of 1 mm, 800 μm, 500 μm, and 300 μm mesh size and allowed for sedimentation. The sediments collected after each filtration were examined for P. metacercariae under the stereomicroscope. The P. westermani-type metacercariae were collected and transferred to screw-capped plastic or glass vials containing 1–2 ml sterile normal saline. The metacercariae were stored at 4°C for up to 1 week for morphological study and experimental animal infection. The normal saline was replaced with fresh saline every day during the period of storage.

Experimental infection of laboratory animal to recover adult flukes

This study was approved by the Institutional Animal Ethics committee of Sikkim Manipal Institute of Medical Sciences, Gangtok, India (approval no. MC/SMIMS/IAEC). Twenty P. westermani-type metacercariae were injected intraperitoneally to a 3-month-old laboratory Wistar rat. The fecal samples of the infected rat were checked (at least twice a week from the 2nd month of infection until autopsy) for the presence of Paragonimus ova by microscopic examination. The necropsy of the rat was performed on day 120 post infection, and two fully matured adult flukes were recovered. The flukes recovered on necropsy were repeatedly washed in sterile normal saline and were kept overnight to allow complete emptying of the excretory bladder. One of the flukes was flattened between two glass slides under increasing pressure/weight and was fixed in 70% ethanol for several days until a well-fixed and flattened mount of about 0.2-mm thickness was obtained. The borax carmine-stained fluke was mounted in Canada balsam, and the whole mount specimen was used for morphological description under the stereomicroscope. The other fluke was preserved in 70% ethanol for future study and reference.


   Results Top


M. lugubris [Figure 1]a were observed to be natural second intermediate hosts of P. heterotremus and P. westermani. The P. westermani-type metacercariae were ovoid and measured 460–565 μm with black “I-” shaped excretory bladder encased within the inner cyst and flanked by the convoluted intestinal tubule [Figure 1]b.
Figure 1: (a) Freshwater crab host Maydelliathelphusa lugubris collected from Churachanpur district. (b) Paragonimus westermani metacercariae from crab host Maydelliathelphusa lugubris. (c) Borax carmine-stained adult worm of Paragonimus westermani

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Morphology of adult flukes

The borax carmine-stained whole-mount specimen showed morphological features characteristic of P. westermani [Figure 1]c. The body measuring 5.2 mm × 3.1 mm (length × width) was elliptical in shape. The oral and the ventral suckers were similar in size. Cuticular spines were observed all over the body surface except around the oral sucker. Cuticular spines were singly spaced with conical pointed tips. The uterus contained many ova and the seminal receptacle contained sperms. The ovaries located on the right of the midline at a level between the uterus and ventral sucker comprised six club-shaped lobes with secondary lobules at the distal ends. The left and right testes located midway between the ventral sucker and posterior end were elongated, slightly smaller than the ovary, and comprised five lobes each.

Morphology of uterine ova

The ova were golden yellow, were oval shaped, and were broader at the anterior end with a distinct operculum. The mean diameters in length and width (maximum position from the operculated end) were 83.5 μm and 49.46 μm, respectively. The eggshell at the opercular region was thicker than the rest.


   Discussion Top


The report of P. westermani metacercariae infection in a new fresh water crab (Alcomon superciliosum) host in Moreh region together with a clinical case of pulmonary paragonimiasis caused by P. westermani[15] prompted us to examine several fresh water crabs from Moreh and its neighboring district (Churachandpur) for P. westermani metacercariae. In our study, M. lugubris (crabs) collected from the mountain streams of Churachandpur were found harboring P. westermani metacercariae. Barythelphusa lugubris (now renamed as M. lugubris) found in Arunachal Pradesh and Meghalaya have been previously reported as second intermediate hosts of P. westermani.[17] In Manipur, A. superciliosum and M. lugubris were found as the natural second intermediate host species of P. westermani. In contrast, Potamiscus manipurensis, a dominant species harboring P. heterotremus, was not found as a permissive crab host to P. westermani. This finding indicates that a variety of fresh water crab species may serve as second intermediate crustacean hosts of Paragonimus species in different geographical areas. The variations in the morphological features of metacercariae within the same species or similarities between different species add difficulties to the species identification process. However, P. heterotremus metacercariae are morphologically distinct from other species and hence are easy to identify. Further, the occurrence of different morphological forms of metacercariae of the same species in a single crab host and/or locality may reflect the differences in the microenvironment within the crab host.[17] In addition, age of the larva and type of the tissue where it encysts can influence the morphological features. Based on the morphological features of adult fluke observed in this study, the species was identified as P. westermani. In addition, the morphological features of the uterine ova such as broader anterior end, distinct operculum/shoulder, and thickened eggshell conformed to the typical features of P. westermani ova. However, the determination of species based only on the morphology of ova would be presumptive, as similar common features may also be observed in the ova of P. heterotremus and P. skrjabini. The possible occurrence of different subtypes within the P. westermani complex or novel Paragonimus species also cannot be ruled out because atypical morphological forms of P. metacercariae are detected from the crab hosts in Manipur and other Northeast states.[17] In this regard, any dispute in the species identification of P. westermani complex can only be conclusively addressed by genetic sequencing of ribosomal rDNA and phylogenetic analysis. However, we did not proceed further to investigate on P. heterotremus as a number of research articles have already been published on the occurrence, morphology, pathobiology, and molecular characterization and its association with human infections in India.


   Conclusion Top


M. lugubris, fresh water crab species, were found as the natural permissive hosts of P. westermani in Manipur region of India. Species identification based on only morphological features is a limitation of this study. Nevertheless, to date, only P. heterotremus and P. westermani were confirmed to be human pathogens in India, and the role of P. skrjabini, P. hueitungensis, and P. macroorchis in human infections is yet to be determined. Further investigations are necessary to identify the role of other crab hosts species as intermediate host of lung flukes in this region.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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2.
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Parihar NS, Shrivastava SN. Bronchial hyperplasia in a tiger (Panthera tigris). Indian J Anim Sci 1988;58:230-3.  Back to cited text no. 4
    
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Sano M, Agrawal MC, Kotwal PC, Gopal R. Paragonimus infection in tigers at Kanha National Park. J Parasitol Appl Anim Biol 1994;3:115-6.  Back to cited text no. 5
    
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Singh TS, Singh YI. Three types of Paragonimus metacercariae isolated from, Potamiscus manipurensis in Manipur. Indian J Med. Microbiol 1997;15:159-62.  Back to cited text no. 6
    
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Singh TS. Occurrence of the lung fluke, Paragonimus hueitungensis in Manipur, India. Chinese Med J (Taipei) 2002;65:426-9.  Back to cited text no. 7
    
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Singh TS. Occurrence of the lung fluke, Paragonimus heterotremus in Manipur, India. Chinese Med Sci J 2003;18:20-5.  Back to cited text no. 8
    
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Singh TS, Singh LD, Sugiyama H. Possible discovery of Chinese lung fluke, Paragonimus skrjabini in Manipur, India. Southeast Asian J Trop Med Health 2006;37:53-6.  Back to cited text no. 9
    
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Shibahara T, Kitagawa H, Singh TS, Singh SM, Singh YI, Terasaki K, et al. The first case of Paragonimus macroorchis C Xhen, 1962 in India. Parasitol Int 1998;47:283-389.  Back to cited text no. 10
    
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Singh TS, Sugiyama H, Achariya R, Devi KR. Morphological and molecular characterization of Paragonimus heterotremus, the causative agent of human paragonimiasis in India. Southeast Asian J Trop Med Public Health 2007;38:82-6.  Back to cited text no. 11
    
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Devi KR, Narain K, Bhattacharya S, Negmu K, Agatsuma T, Blair D, et al. Pleuropulmonary paragonimiasis due to Paragonimus heterotremus: Molecular diagnosis, prevalence of infection and clinicoradiological features in an endemic area of northeastern India. Trans Roy Soc Trop Med Hyg 2007;101:786-92.  Back to cited text no. 12
    
13.
Prasad PK, Tandon V, Biswal DK, Goswami LM, Chatterjee A. Phylogenetic reconstruction using secondary structures and sequence motifs of ITS2 rDNA of Paragonimus westermani (Kerbert, 1878) Braun, 1899 (Digenea: Paragonimidae) and related species. BMC Genomics 2009;10 Suppl 3:S25.  Back to cited text no. 13
    
14.
Devi KR, Narain K, Agatsuma T, Blair D, Nagataki M, Wickramasinghe S, et al. Morphological and molecular characterization of Paragonimus westermani in northeastern India. Acta Trop 2010;116:31-8.  Back to cited text no. 14
    
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Singh TS, Hiromu S, Devi KR, Singh WA. First case of Paragonimus westermani infection in a female patient in India. Indian J Med Microbiol 2015;33 Suppl:156-9.  Back to cited text no. 15
    
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Takeda M, Sugiyama H, Singh TS. Some freshwater crabs from northeast India bordered on Myanmar. J Teikyo Heisei Univ 2012;23:199-213.  Back to cited text no. 16
    
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Iwagami M, Rajapakse RP, Paranagama W, Okada T, Kano S, Agatsuma T. Ancient divergence of Paragonimus westermani in Sri Lanka. Parasitol Res 2008;102:845-52.  Back to cited text no. 17
    


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