CHAPTER – I

STATUS AND PREVALENCE OF

CRUSTACEA-BORNE TREMATODE

ZOONOSES IN THE REGION

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INTRODUCTION

Zoonoses are the diseases which are naturally transmitted between animals

and humans. Parasitic zoonoses are well recognized and important public health

problems both in developing and developed countries. The burden of diseases caused

by food-borne pathogens (microbial, protozoal and macrobial) remains largely

unknown. Emerging microbial and protozoan zoonoses are major causes of diarrhoeal

disease in humans worldwide (Koopmans and Duizer, 2004; Smith et al., 2007). A

wide spectrum of helminthic infections is transmitted to humans by contamination of

food and water (Lloyd and Soulsby, 1998; Eckert et al., 2000; Muller, 2001; Vander

Hoek et al., 2003). The human behavioural patterns including habits, customs,

traditions and socio economic practices are closely related to such infections.

Globalization of the food supply, increased international travel, increase in the

population of highly susceptible persons, and change in culinary habits are some

factors associated with the increased diagnosis of food-borne parasitic diseases

worldwide (Dorny et al., 2009). Zoonotic parasitic diseases are transmitted to humans

either by ingesting environmentally robust transmissive stages (spores, cysts, ova,

larval and encysted stages) or by eating raw or undercooked `meat containing

infective tissue stages. Humans can be final, intermediate or paratenic (maintenance)

or accidental hosts. While the transmissive stages of some of these zoonoses can be

transmitted directly (e.g. by animal-human contact or through contact with

contaminated faeces, soil and garbage), they can also be transmitted through

contaminated water and food. A variety of infective tissue parasite stages are

responsible for transmitting meat- and fish-borne zoonoses. Eating raw, undercooked,

cured, smoked, salted, pickled or air-dried meat can increase the risk of contracting

food-borne parasite zoonoses, especially when the preservation treatment is

inadequate. Food-borne trematode infections, acquired through eating raw,

improperly cooked or processed freshwater fish, shell-fish, crabs, or unwashed or

inadequately washed vegetables have been well recognized as a public health problem

as evident from a study carried out under the “Southeast Asian Ministries of

Education Organization Regional Tropical Medicine and Public Health Project”

(SEAMEO-TROPMED) (Eduardo, 1991). Clonorchiasis, paragonimiasis, fascioliasis,

fasciolopsiasis and other intestinal trematodiases are the most important diseases

3

contracted, and strategies to control food-borne trematode infections have been

identified (WHO, 1995; Theresa et al., 2000). Many crustacea including crabs and

crayfishes themselves constitute an important food resource in hill tribes. Freshwater

crabs are also consumed for purported medicinal and tonic properties, including

treatment of stomach ailments and physical injuries (Dai, 1999; Darren et al., 2008).

Crabs also play an important role as the second intermediate host in the life cycle of

several trematode parasites and serve as a biological vector for transmission of their

infective stages to the final host. Besides, these crustaceans have also been reported as

potential vectors for cestode and acanthocephalan parasites from many parts of the

globe (Bratty et al., 1985; Thompson, 1985; Kuris and Gurney, 1997; Torchin et al.,

2001).

Crustacea-, snail- and water (aquatic vegetation) - borne trematodiases:

The lungflukes of the genus Paragonimus have been known as one of the most

important zoonotic parasites causing paragonimiasis in man, for which crabs serve as

the transmitting agents. Adult worms mature in capsules in the lungs and deposit ova,

which can be found on expectoration. The disease is often misdiagnosed as

tuberculosis due to overlapping clinical symptoms including chest pain, cough,

haemoptysis and confusing radiological findings on chest radiography (Toscano et al.,

1995). The parasite can migrate to several other vital tissues including the brain along

nerve sheaths (Kusner and King, 1993). Humans, dogs, cats and wild carnivores serve

as definitive hosts and are infected by eating undercooked crabs, crayfish or shrimp.

Crabs which are sometimes pickled in wine or brine, are a major source of

Paragonimus infection (Calum and Macpherson, 2005). More than 50 species are

known to infect different mammalian hosts throughout the world and approximately

15 species are known to infect humans (Bunnag and Harinasuta, 1985). The best-

known species is Paragonimus westermani (Kerbert 1878) Braun 1899 - a human

parasite that can undergo development in as many as 16 different snail species and 50

crustacean species (Blair et al., 1999b). Paragonimiasis, affecting more than 22

million people worldwide, is due to infection with P. westermani, P. miyazakii, P.

skrjabini and P. heterotremus in Asia, P. africanus and P. uterobilateralis in Africa,

and P. mexicanus and other species in Latin and South America. Countries with large

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numbers of cases include Asia, Nigeria, Cameroon, Peru and Ecuador (Blair et al.,

1997, 2005).

In Indian context, Chandler and Read (1961) indicated Bengal, Assam and

some other parts of the country as endemic foci of human paragonimiasis. In India,

human paragonimiasis was first reported in Manipur by Singh et al. (1982), followed

by more reports in later years (Singh et al., 1986, 1993, 2004). The metacercarial

stages recovered from the freshwater crab, Potamiscus manipurensis, in

Churachandpur area were grown to adult stage in experimental dog hosts and based

on morphological criteria the fluke was confirmed to be Paragonimus hueit’ungensis

Chung, 1977- a species originally reported from China (Singh, 2002). Two more

species of Paragonimus have been reported as occurring in Manipur- P. skrjabini

from the same crab host (P. manipurensis) and P. heterotremus from Indochinamon

manipurensis (Singh et al., 2006, 2011).

Suspected foci of human paragonimiasis have also been reported from some

locales in Arunachal Pradesh (Narain et al., 2003). In Changlang district of Arunachal

Pradesh, during a community based survey it was reported that 95% of the local

inhabitants had the habit of consuming crabs and crayfishes in half cooked or in raw

form. To investigate the possible effect of human infection, stool and sputum samples

were also collected from suspected foci of infection; of these, two stool samples were

found positive for Paragonimus eggs. Two species of crustacean hosts, Barytelphusa

lugubris mansoniana and Sartoriana spinigera, were surveyed from the same locality,

but only the former were found positive for metacercarial infection; up to 1000

metacercariae were recovered from a single crab host. The species was identified as

P. heterotremus, the species implicated in human infection (Narain et al., 2003). In

subsequent studies based on surveys carried out in Kharshang and Miao regions of the

same district, the occurrence of P. westermani was also reported (Tandon et al., 2007;

Devi et al., 2010). In suspected endemic areas of the region, P. heterotremus is

reported to be implicated in human pleuropulmonary paragonimiasis (Devi et al.,

2007).

Apart from Paragonimus, many other trematodes, members of the family

Microphallidae in particular, utilize crabs as the intermediate host (Anantaraman and

Subramoniam, 1976; Heard and Overstreet, 1983; Pung et al., 2002). The family

Microphallidae Ward, 1901 indicates a large assemblage of small sized (usually <

1mm) digenean taxa (that represent more than 160 species under 28 genera arranged

5

in 10 subfamilies), which characteristically harbour the intestine of all groups of

vertebrates, mainly ducks (Martorelli et al., 2004) and mostly rodents among

mammals (Deblock, 1971, 2008). From amongst all these microphallids, only one,

viz. Spelotrema brevicaeca is reported to infect humans (Fried et al., 2004). The

infective metacercarial stages of microphallid flukes commonly occur in Crustacea

and are known to undergo extensive organogenesis in these intermediate hosts

(Caveny and Etges, 1971; Heard and Overstreet, 1983). Many workers have made

significant contributions to the studies of microphallid life histories (Cable and

Hunninen, 1940; Stunkard, 1957, 1958; James, 1968; Deblock, 2008). Most of the

adult forms have been recovered from aquatic birds, which get infected probably by

consumption of infected crustaceans. The taxon-rich digenean superfamily

Microphalloidea Ward, 1901 is an assemblage of 18 or more families, the taxonomic

history of which is rather complex and not free from debatable opinions regarding the

placement of various taxa; like most digenean trematodes microphallid taxa also

encompass several species/genera with little morphological differentiation (Bray et

al., 2008).

In addition to the above mentioned fluke infections, infective stages of several

other trematodes (eg. the fasciolid, amphistomid and echinostomid species), may

occur in the same acquatic environment in consonance with the crustacean

transmitters of trematodiases. The amphistome Gastrodiscoides and the echinostomid

fluke Artyfechinostomum are among the trematodes that have a zoonotic potential.

Gastrodiscoides hominis Lewis et McConnell, 1876 is commonly found in caecum

and colon regions of pig and human, where pig is a normal host species (Ahluwalia,

1960; Kumar, 1980; 1999; Mas-Coma et al., 2005), the infection in humans being a

rare occurence (Dada-Adegbola et al., 2004). The exact life cycle is unknown but

probably similar as in other species of Gastrodiscidae, involving aquatic vegetation as

the second intermediate environment that is used for the encystment of the

metacercarial infective stage (Zablotski, 1964; Dutt and Srivastava, 1972; Mas-Coma

et al., 2007). Gastrodiscoidiosis has symptoms similar to diarrhoea and, if untreated,

might kill the patient, mostly children (Kumar, 1980). G. hominis has a wide

distribution throughout India including the states of Assam, Bengal, Bihar, Uttar

Pradesh, Madhya Pradesh and Orissa (Shrivastav and Shah, 1970; Murty and Reddy,

1980). Buckley (1964) reported a high prevalence of G. hominis in humans, mostly

children (around 41%), in Kamrup District of Assam. In a later study, carried out in

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Meghalaya, this parasite was shown to have a pattern of seasonal prevalence (Roy and

Tandon, 1992). Apart from India, G. hominis is widely distributed in countries like

Pakistan, Burma, Thailand, Vietnam, Philippines, China, Kazakhstan, and Russia

(Buckley, 1939; Ahuwalia, 1960; Kumar, 1980; Harinasuta et al., 1987; Yu and Mott,

1994; Ivanov and Semenova, 2000).

Artyfechinostomum sufrartyfex Lane, 1915 (synonyms- A. malayanum Leiper,

1901 Railliet, 1925; Euparyphium malayanum Leiper, 1911; Testifrondosa cristata

Bhalerao, 1924; Paryphostomum mehrai, Jain, 1957; Neoartyfechinostomum subhrai

Agarwal, 1963; Rao et Niphadker, 1963) is an echinostome intestinal fluke of pigs

that causes echinostomiasis. The first human infection of A. sufrartyfex was reported

in an 8-year old girl in Assam (Lane, 1915). Although echinostomiasis occurs world-

wide, most human infections have been reported from East and Southeast Asia.

Despite being rare, at least 19 species of echinostomes from 8 genera have been

reported in humans from China, Indonesia, Japan, Korea, Malaysia, Russia, Taiwan

and Thailand (Wanachiwanawin and Ungkanont, 2001). In India, the occurrence of A.

sufrartyfex has been reported from the states of Andhra Pradesh, Assam, Bihar, Tamil

Nadu, Uttar Pradesh and West Bengal (Beaver et al., 1984; Anonymous, 2005).

Another species of Artyfechinostomum, A. oraoni has been found to be of endemic

occurrence among the Oraon tribes of West Bengal (Bandopadhyay et al., 1989; Maji

et al., 1995). The human infection is acquired by ingestion of poorly cooked

freshwater fishes, prawns, crabs, molluscs and tadpoles (Adams and Motarjemi,

1999). Like most trematode eggs, identification of echinostome eggs as well as the

adult is difficult because of morphological similarity between biologically different

taxa and historical nomenclatural problems (Kostadinova and Gibson, 2000). Collar

spination, the first intermediate host (snail species) and geographical distribution are

the main diagnostic characters utilized for identification of the species; however,

sometimes these characters are also of limiting value (Morgan and Blair, 1995).

The present study was aimed at studying the trematode infections prevailing

among the edible crab species, the potential intermediate hosts for digenetic flukes in

selected localities in the region, Assam and Meghalaya in particular. For identifying

the species implicated in infection in the region, morphological criteria of the intra-

molluscan stages and metacercaria recovered were used including surface fine

topography. While the infective metacercaria stage is harboured in the crab host, the

preceding larval stages (sporocyst, redia, cercaria) develop in the snail (first

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intermediate) hosts. Therefore, in order to ascertain the prevalence status of these

infections, snail hosts were also included in the survey study.

MATERIALS AND METHODS

1. Survey of intermediate hosts

With reference to the previously recorded cases, the survey was carried out in some

selected pockets of Arunachal Pradesh, Assam, Meghalaya, Nagaland and Tripura

wherein the native populations use crabs and snails as part of their food.

(i) Crabs: In rural markets of Northeastern India, crabs are commonly sold as a food

item. In rural localities and countryside of the region, eating of crabs is a common

food practice among the natives. Therefore, the collection and survey of crabs were

restricted to a selected few sites, which are shown in the map of the region (Fig. 1.1).

A total of 1548 crustacean hosts representing 3 species were examined for the

recovery of metacercariae, if any from them (Table 1.1). Naturally infected freshwater

crabs were periodically collected, mostly from mountain streams, paddy fields and

local markets in suspected foci of infection (Fig. 1.2, 1.3a-c). Further collections were

continued in only those locations where the crustaceans were found positive for

infection.

(ii) Snails: These gastropod molluscs, serving as the first intermediate host in the life

cycle of most trematodes, harbour in their soft tissues the pre metacercarial

developmental stages like the sporocyst and redia, with the latter giving rise to many

cercariae that emerge from the snail and invade the second intermediate host. Keeping

this fact in mind, snails were collected from all those aquatic bodies and localities

wherever the crab hosts were suspected to be positive for metacercarial infections

(Fig. 1.3d-g). Hundreds of snail specimens representing 4 genera with one species

each were examined for recovery of intra-molluscan stages of digenetic trematodes

(Table1.2).

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Fig. 1.1. Map of Northeast India showing the collection sites

1= Changlang District - Miao (Altitude 429 meters)

2= Nagaon District - Kaliabor (Altitude 69 meters)

3= Kamrup District - Boko (Altitude 65 meters)

4= West Garo Hills District - Tura (Altitude 698 meters)

5= East Khasi Hills District - Nongrim (Altitude 578 meters)

6= Peren District - Jalukie (Altitude 1395 meters)

7= Mokokchung District- Changki (Altitude 1345 meters)

8= South Tripura District - Udaipur (Altitude 27 meters)

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Fig. 1.2. A collection site in Garo Hills and crabs for sale in the rural market

area

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Table 1.1. Surveyed areas for crab hosts for recovery of metacercarial stages

Sl. No Locality Species Status of infection

1 Arunachal Pradesh

Miao Barytelphusa lugubris mansoniana +

Sartoriana spinigera _

2 Assam

Nagaon B. l. mansoniana +

Kamrup Lobothelphusa fungosa _

3 Meghalaya

Tura B. l. mansoniana +

Nongrim B. l. mansoniana and _

S. spinigera _

4 Nagaland

Mokokchung B. l. mansoniana _

Peren B. l. mansoniana _

5 Tripura

Udaipur B. l. mansoniana _

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Table 1.2. Surveyed areas for snail hosts for recovery of intramolluscan

trematode larval stages

Sl. No Locality Species Status of infection

1 Arunachal Pradesh

Miao Brotia costula +

2 Assam

Nagaon Bellamya bengalensis +

3 Meghalaya

Tura Paludomus loricatus +

B. bengalensis +

Melanoides tuberculata _

Nongrim M. tuberculata _

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Fig. 1.3. Crab and molluscan species surveyed

a. Barytelphusa lugubris mansoniana (Henderson)

b. Lobothelphusa fungosa (Alcock)

c. Sartoriana spinigera (Wood-Manson)

d. Paludomus loricatus (Reeve)

e. Bellamya bengalensis (Kobelt)

f. Melanoides tuberculate (Muller)

g. Brotia costula (Rafinesque)

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2. Recovery of the parasite material

(i) From Crabs: Metacercariae were isolated from the muscles of the crustacean host

by digestion technique. The crabs were cut into small pieces with the help of scissors

and digested by overnight incubation at 37oC in the artificial gastric juice

[Composition: HCl conc. (35 – 37%) 7-10 ml, distilled water – 1000 ml, pepsin

(1:10000) – 6 g].

The digested materials were filtered through mesh wire sieves and the

filterable sediments were washed repeatedly with tap water in order to get a clearer

supernatant. The sediment was examined for metacercariae under a dissecting

stereoscopic microscope.

(ii) From Snails: Snails to be examined were kept in a water-filled glass beaker and

exposed to sunlight for half-to-one hour prior to recovery of intra-molluscan stages.

Exposure to sunlight stimulates the shedding of cercariae from the infected snail into

the water. The shell of the mollusc specimen was broken open and its soft tissues

teased in a petri dish containing water. The contents were examined under a

stereoscopic microscope for recovery of sporocyst, redia or cercaria stages, which

were picked up and duly processed for further morphological studies.

3. Light microscopy (LM)

Freshly recovered metacercariae from crab hosts and intra-molluscan larval stages

were washed in 0.7% saline solution and narcotized with few drops of 70% ethyl

alcohol. The metacercariae were gently excysted using a fine needle, flattened

between a slide and a cover glass, fixed in 70% ethyl alcohol and stained with Borax

carmine and Mayer‟s carmellum stains, followed by dehydration through usual

dehydration media, i.e., ascending grades of ethyl alcohol, clearing in Methyl

benzoate and mounting in Canada balsam using standard protocols. For histological

study, serial sagittal sections of the fresh frozen material were also cut at a thickness

of 8-10 µm, using Leica CM 1850 cryotome.

The intra-molluscan larvae were also duly processed for whole mount

preparations following the standard protocol. All the prepared permanent slides were

observed and studied using the various optical systems available in the laboratory-

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Wild M5APO stereo microscope, vision analyser, Zeiss image analyser, Leica DM

1000 image analysis system and Leitz Ortholux-2 research microscope.

Measurements of the specimens were taken using stage and ocular micrometers

and/or morphometric software in the image analyzer.

For identification of the parasite standard reference works were followed

(Yamaguti, 1971; Bray et al., 2008).

4. Scanning electron microscopy (SEM)

The isolated metacercariae were fixed in 2.5% glutaraldehyde in 0.1M sodium

cacodylate buffer for 6 hour at 4°C, washed in phosphate buffered saline and

dehydrated with ascending grades of acetone to pure dried acetone. The specimens

were then treated with Tetra methyl silane (TMS) in lieu of critical point drying

following Roy and Tandon (1991). The gold-coated specimens were observed using

LEO 435 VP scanning electron microscope at electron-accelerating voltages ranging

between 10 and 20 kV.

5. Analysis of prevalence data

The following parameters were used to analyze the data following Bush et al. (1997):

(i) Prevalence - the number of individuals in a population estimated to be infected

with a particular species of parasite (usually expressed in percentage).

(ii) Range - minimum to maximum number of individuals of a particular parasite

species in infected hosts in a sample.

(iii) Mean intensity - the average intensity, i.e., the total number of parasites of a

particular species found in a sample divided by the number of hosts infected; and

(iv) Abundance - the total number of individuals of a particular parasite species in a

sample of a particular host species divided by the total number of hosts (including both

infected and uninfected) of that species examined.

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OBSERVATIONS/RESULTS

1. Larval trematode infection among crabs and molluscan hosts in

the suspected areas:

(i) In crab hosts: Of the three edible crab species surveyed from various localities in

Arunachal Pradesh, Assam, Meghalaya, Nagaland and Tripura only one, i.e.,

Barytelphusa lugubris mansoniana was found to be naturally infected with

metacercarial forms. The distribution data of the parasites recovered are presented in

Table 1.3. The crab collected from only three localities, namely- Miao (in Changlang

District of Arunachal Pradesh), Tura (in West Garo Hills District of Meghalaya), and

Nagaon (in Assam) were found positive for metacercarial infection; B. l. mansoniana

from all other collection sites (in Nagaland and Tripura) did not harbour any infection.

The collection comprised metacercariae of three morphologically distinct

types. The type - I and type - II metacercariae both had characteristics of juvenile

Paragonimus flukes, whereas the type - III metacercaria showed a typical of

microphallid flukes. As a rare instance of occurrence, the type - II metacercaria was

recorded only once despite repeated surveys of crustacean hosts during the entire

period of the study. Though representing a paragonimid species, this metacercaria was

distinctly differentiable from the type - I metacercaria. The latter, also a paragonimid,

was recovered only from Barytelphusa crabs from the foci of infection in Arunachal

Pradesh. A high prevalence (85%) of the type - I metacercaria was recorded in the

study site. The crabs from the Tura region showed a very high prevalence (91.05%) of

infection of type - III metacercaria. These microphallid metacercariae were recovered

in high numbers (ranging between 53-285) per infected host and showed a high mean

intensity as well. In comparison, the crabs surveyed from Nagaon region also

harboured the similar infection, though with a low prevalence and low intensity of

infection.

(ii) In snail hosts: With a view to recovering the possible infection of larval

trematodes from the molluscan host, snails were also screened only in those areas

where crabs showed positive for the metacercarial infection. The details of molluscan

hosts examined from the various localities and the status of infection among them is

16

depicted in Table 1.4. Of the four species of snails, prevalent in the study site, three,

viz. Paludomus loricatus, Bellamya bengalensis and Brotia costula were found

infected with trematode larvae; Melanoides tuberculate did not show any infection.

While P. loricatus from Meghalaya and B. costula from Arunachal Pradesh harboured

both redia and cercaria stages (Fig. 1.4a,b), B. bengalensis from Assam harboured

only cercaria (Fig. 1.4c). In morphological identification, the cercarial stages were

revealed to be of two types – one a distome leptocercous (with long slender tail) and

opthalmate cercaria having a pair of eye spots (Fig. 1.4b), and the other an

amphistome cercaria having its ventral sucker located at the base of the fore body

(Fig. 1.4c).

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Fig. 1.4. Intra-molluscan stages recovered

a. Redia

b. Leptocercous cercaria

c. Amphistome cercaria

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Table 1.3. Status of metacercarial infection in crustacean hosts

Locality Crab species Metacercaria Number

examined

Numbers

infected

Prevalence

(%) Range

Mean

Intensity Abundance

Arunachal Pradesh

Miao Barytelphusa lugubris Type I (paragonimid) 84 71 85% 11-182 54.33 45.92

mansoniana,

40 _ _ _ _ _

Sartoriana spinigera

Assam

Nagaon B. l. mansoniana Type III (microphallid) 108 17 15.70% 05-023 10.47 1.64

Kamrup Lobothelphusa fungosa _ 115 _ _ _ _ _

Meghalaya

Tura B. l. mansoniana, Type III (microphallid) 917 835 91.05% 53-285 74.7 68.02

Type II (paragonimid) 7 3 43% 02-05 3.33 1.4

Nongrim B. l. mansoniana, _ 43 _ _ _ _ _

S. spinigera _ 27 _ _ _ _ _

Nagaland

Mokokchung B. l. mansoniana _ 95 _ _ _ _ _

Peren B. l. mansoniana _ 81 _ _ _ _ _

Tripura

Udaipur B. l. mansoniana _ 31 _ _ _ _ _

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Table 1.4. Larval trematodes in molluscan hosts examined from the focal sites of metacercarial infections in crabs

Locality surveyed Snail species Numbers examined Numbers infected (%) Trematode larva type

Arunachal Pradesh Miao Brotia costula 527 50 (9.48%) Redia/ Cercaria

Assam

Nagaon Bellamya bengalensis 335 65 (19.4%) Cercaria

Meghalaya

Tura Paludomus loricatus 362 70 (19.33%) Redia/ Cercaria

B. bengalensis 308 55 (17.85%) Cercaria

Melanoides tuberculata 259 _ _

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2. Morphology of metacercariae

(i) Metacercaria type - I (paragonimid metacercaria - I):

The metacercarial cyst is oval in shape with a smooth surface. The body

surface of the excysted metacercaria is covered with numerous pointed tegumentary

spines. The tegument in the circum-oral region has a dense aggregation of small

spines that are arranged in several circular rows. On the rim of the oral sucker and

adjacent area, a few dome-shaped papillae are present in a random fashion. The

tegument of the ventral sucker and its surrounding region and that of the general body

surface also has a dense spination. The excysted metacercaria is elongated, 0.815 mm

x 0.492 mm in size, the ventral sucker is situated somewhat pre-equatorially, larger

than the oral sucker; the intestinal caeca are long and extend up to the posterior end of

the body; the conspicuous excretory bladder extends medially in the intercaecal space.

The metacercarial juvenile form is identified as Paragonimus westermani (Fig. 1.5a-

c).

(ii) Metacercaria type - II (paragonimid metacercaria - II):

From the crab species B. l. mansoniana,, another paragonimid type of

metacercaria was also recovered, though in very low number (2-5). These

metacercariae are much smaller in size than the type – I. The metacercarial cysts are

oval in shape, measuring 0.058 mm in length and up to 0.048 mm in maximum width;

the metacercarial cyst has a single layered thin wall. The excysted metacercaria is

described below (Fig. 1.6, a-f).

Description (based on 5 metacercariae): The excysted metacercaria has an

elongated body, with an average length of 0.09 mm and 0.034 mm in width. The fluke

inside fills the entire space within the cyst. The ventral sucker in the juvenile fluke is

situated pre-equatorially and is almost equal to or slightly larger than the oral sucker.

The intestinal caeca are long and extend up to the posterior extremity of the body. The

excretory bladder is medially extended between the caeca till the posterior end.

These characters are in conformity with the morphology of paragonimid

metacercaria stages. Therefore, the metacercarial type-II form represents the genus

Paragonimus.

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Fig. 1.5. Metacercaria type – I. LM view

a. Encysted metacercaria;

b. Metacercaria excysting and emerging out of the cyst

c. Excysted metacercaria

Fig. 1.6. Metacercaria type – II. LM view

a. Encysted metacercaria

b. Metacercaria breaking open the cyst wall

c. Excysted metacercaria with remnant of the cyst covering

d. Enlarged view of the anterior half of body

e. Mid body region, with ventral sucker

f. Posterior portion of body in a closer view

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(iii) Metacercaria type - III ( = microphallid metacercaria):

The collection comprised a large number of type - III metacercarial cysts (53-

285) recovered from the infected crabs Barytelphusa lugubris mansoniana.

The cysts are elliptical in shape and have a prominent thick wall, which is

composed of two layers, the outer layer being thick but transparent. The excysted

metacercaria is described below.

Family Microphallidae Ward, 1901

Subfamily Microphallinae Ward, 1901

Genus Microphallus Ward, 1901

Description (based on 10 excysted specimens; Figs. 1.7, 1.8): Most of the

adult characters prominent; body pyriform in shape, minute in size (1.035 mm in

length, 0.655 mm in maximum width). Oral sucker subterminal; ventral sucker single

(not two), well developed, post equatorial in position. Prepharynx conspicuous;

pharynx small, short, muscular; oesophagus long, bifurcating in mid body region;

intestinal caeca short, ending in region at level of ventral sucker, pretesticular. Testes

symmetrical, located on either side of ventral sucker; cirrus pouch present, curved in

shape; cirrus (= phallus) well developed, conical, occupying entire genital atrium.

Genital atrium opening near by ventral sucker. Ovary located on right side of ventral

sucker or slightly overlapping right testis, oviduct emerging from mid posterior part

of ovary; uterus forming ascending and descending loops between testes and

excretory vesicle, containing numerous eggs. Vitelleria commencing from level of

testes, extending up to excretory vesicle; vitelline glandular cells arranged in two

groups- one with eight lobes on right, other with seven on left. Excretory vesicle „V‟

shaped, excretory pore terminal.

The morphometric measurements (mm) of the body and its organs are

provided in Table 1.5.

The SEM observations revealed the fine surface topography of the

metacercaria (Fig. 1.9). The metacercarial cyst has a smooth surface. The excysted

metacercaria is oval in shape and the entire body surface is covered with numerous

spines, the latter having digitated projections directed posteriorly; numerous papillae

abound in the circum-oral sucker region, where they appear to be randomly scattered

and not showing any pattern.

23

Table 1.5. Morphometric measurements (in mm) of metacercaria type - III

Sl. No Characters Range Mean S.D.

1 Length of body 0.943-1.173 1.035 ±0.099

2 Maximum width of body

(at level of ventral sucker) 0.598-0.713 0.655 ±0.047

3 Oral Sucker: Length 0.042-0.09 0.066 ±0.017

Breadth 0.06-0.09 0.073 ±0.013

4 Ventral sucker: Length 0.087-0.096 0.09 ±0.003

Breadth 0.069-0.09 0.084 ±0.008

5 Location of ventral sucker

from anterior end 0.45-0.648 0.522 ±0.078

6 Length of Pharynx 0.024-0.032 0.028 ±0.027

7 Length of Oesophagus 0.225-0.396 0.317 ±0.063

8 Testes: Length 0.099-0.159 0.133 ±0.019

Breadth 0.039-0.09 0.054 ±0.016

9 Ovary: Length 0.168-0.198 0.176 ±0.013

Breadth 0.108-0.135 0.122 ±0.014

10 Cirrus Sac: Length 0.36-0.441 0.039 ±0.029

11 Eggs: Length 0.016-0.018 0.016 ±0.0008

Breadth 0.008-0.01 0.008 ±0.0007

24

Fig. 1.7. Metacercaria type – III. Whole mount

25

Fig. 1.8. Metacercaria type – III. LM view

a. Encysted metacercaria

b. Excysted metacercaria, whole mount, ventral view

c. Eggs in utero

d, e, f. Serial sagittal sections confirming the presence of a single

ventral sucker in the juvenile fluke

26

Fig. 1.9. Metacercaria type – III. SEM view

a. Metacercarial cyst

b. Excysted metacercaria

c. Oral sucker with papillae, closer view

d. Body spination

27

DISCUSSION

1. Prevalence status of trematode larval stages

During the field surveys carried out in different suspected foci of lungfluke

infection, the edible crab, Barytelphusa lugubris mansoniana was found to be

dominantly prevalent in the State of Arunachal Pradesh and Meghalaya, whereas in

Assam, in addition to this species, Lobotelphusa fungosa and Sartoriana spinigera

also abound. S. spinigera is available in Arunachal Pradesh as well. Of these three

crab species surveyed, only Barytelphusa crabs were found to be infected with three

types of metacercariae. In case of the study area in Arunachal Pradesh, the

Barytelphusa crab species were found to harbour metacercariae that were quite

different from those recovered from foci in Meghalaya and Assam and a high

prevalence (85%) of these metacercariae (type - I) was recorded from this

mountainous region. The metacercaria type - II, which in morphological characters

revealed conformity to paragonimid flukes, were recovered in very low numbers, just

2-5 per crab host. It was noticed that Barytelphusa crabs from Tura locality were

bearing yet another type of metacercarial infection, i.e., type - III was 91.05% and the

intensity high in crabs collected from Meghalaya; up to 285 metacercariae were

recovered from a single crab host. In Assam, however the intensity of infection was

rather low (15.70%, with maximum 23 metacercariae recovered from a single host).

The type - III forms revealed the characteristics of microphallid flukes.

In previous studies carried out with respect to Paragonimus infection in crabs,

Potamiscus manipurensis was reported to harbor metacercariae of Paragonimus spp

in Senapati District of Manipur (Singh and Singh, 1997). Later, Narain et al. (2003),

Tandon et al. (2007) and Devi et al. (2010) also reported the prevalence of

Paragonimus infection in B. lugubris crabs from Arunachal Pradesh and Meghalaya

respectively. Approximately 52% of children in Arunachal Pradesh (age ≤15 years)

were serologically found positive, of which 40% were expectorating Paragonimus

eggs in their sputum (Devi et al., 2010).

28

2. Morphology

Significant work has been done on metacercarial morphology of several

species of Paragonimus, namely P. skrjabini, P. iloktsuensis, P. ohirai, P.

pulmonalis, P. westermani, P. miyazaki, P. mexicanus, P. heterotremus (He et al.,

1982; Aji et al., 1984; Li et al., 1987; Higo and Ishi, 1987; Tongu et al., 1987;

Sugiyama et al., 1990; Jiang and Xia, 1993; Sugiyama et al., 2001) and microphallid

forms, viz. Microphallus abortivus from Ireland (Saville and Irwin, 1991), M.

sabanensis from Venezuela (Diaz et al., 2004); M. breviatus from Iceland (Kirill et

al., 2007); tegumental ultrastructure of Gynaecotyla squatarolae, Microphallus

koreana and Probolocoryphe uca has also been described (Guk et al., 2008; Lim et

al., 2008; Wafa et al., 2010).

On the basis of morphological criteria, the metacercarial type - I was

identifiable as Paragonimus westermani. The infection of P. westermani, a potential

zoonosis, has been reported to be dominantly prevalent in Barytelphusa spp. in

Arunachal Pradesh (Tandon et al., 2007). Though Devi et al. (2010) have reported the

occurrence of P. westermani in crabs of Meghalaya region, in the present study, this

species of lungfluke was not encountered from foci in even once during the entire

period of study.

With regard to the type - II metacercarial form, the main characters observed

were small, oval shaped metacercarial cyst having a thin and single layered cyst wall;

in the juvenile fluke the ventral sucker is located at the mid body level and

conspicuous intestinal caeca extend up to the posterior end of the body. These

characters are in conformity with the metacercarial stage of Paragonimus species

(Waikagul et al., 1989). In earlier studies from the Northeastern states, several species

of Paragonimus were recorded from different crab hosts; these include P.

hueit’ungensis and P. skrjabini from Potamiscus manipurensis; P. heterotremus from

Indochinamon manipurensis, all in Manipur (Singh et al., 2002, 2006, 2011); P.

heterotremus and P. westermani from B. l. mansoniana in Arunachal Pradesh (Narain

et al., 2003; Tandon et al., 2007); and P. westermani from B. l. mansoniana in

Meghalaya (Devi et al., 2010). But the present metacercarial form does not resemble

any of the above described species. On the basis of morphological characters the

present metacercaria (type - II) resembles Paragonimus macrorchis Chen, 1962, a

lungfluke species originally described from the crab host Potamon smithianus in

29

Thailand. However, to confirm the species status, the molecular taxonomic approach

is very much essential in this regard.

The type - III metacercaria in the present study bears the characters, namely

spiny body surface, ventral sucker at the middle third of the body, prepharynx distinct,

pharynx well developed, oesophagus moderately long, caeca short (may or may not

reach the level of testes); testes symmetrical, equatorial or at the posterior half of the

body, cirrus pouch more or less curved, ovary submedian and lateral to ventral sucker,

uterine coils confined to hind body, eggs small and excretory vesicle „V‟ shaped. In

having these features of morphology, particularly the precocious sexual development

while still in larval stage, the present form belongs to family Microphallidae Ward,

1901 [syn. Maritrematidae Nicoll, 1907]. As per Deblock (2008), there are nine

subfamilies under the family, which includes Microphallinae Ward, 1901;

Levinseniellinae Stiles & Hassall, 1901; Maritrematinae Nicoll, 1907;

Gynaecotylinae Guschanskaya, 1952; Pseudolevinseniellinae Tsai, 1955;

Basantisiinae Yamaguti, 1958; Sphairiotrematinae Deblock & Ky, 1966;

Androcotylinae Deblock & Heard, 1970; and Endocotylinae Deblock, 1971. After

considering all the characters, following the classification of Yamaguti (1971) and

Deblock (2008) the larval trematode form described herein comes under the

subfamily Microphallinae and the tribe Microphallini. There are six genera under the

tribe; these are: Microphallus (Microphallus) Ward, 1901 [Syns. Spelotrema

Jagerskiold, 1901; Monocaecum Stafford, 1903; Paraheterophyes Afanassief, 1941;

Carneophallus Cable & Kuns, 1951; Pseudocarneophallus Yamaguti, 1971;

Bulbovitellus Yamaguti, 1971; Feminacopula Ke, Liang & Yu, 1987; Microphallus

(Spelophallus) Jagerskiold, 1908]; Megalophallus Cable, Connor & Balling, 1960;

Atriophallophorus Deblock & Rose, 1964; Megalophalloides Ching & Ibanez, 1976;

Megistospermaticus Deblock & Canaris, 1992; and Rhyncostophallus Deblock &

Canaris, 1997. The present form comes close to the genus Microphallus in several

diagnostic characters viz. body linguiform, flattened; ventral sucker post equatorial,

without superficial veil; oesophagus medium sized; caeca short, divergent at the level

of ventral sucker; testes ovoid, post-ovarian and symmetrical; cirrus (phallus) atrial,

muscular and fleshy; genital atrium simple, enveloping the phallus closely; ovary

directly in front of right testes; uterus post caecal and coiled around the testis;

vitellaria formed of two relatively large clusters, each comprising 7-8 follicles,

vitelline ducts medium-sized, arched; and excretory vesicle short, post testicular, V-

30

shaped. In SEM observations the metacercarial cysts under the present study revealed

a smooth surface contour, whereas the excysted juvenile fluke has a spiny tegument

and numerous papillae in the region surrounding the oral sucker. Surface fine

topography of encysted and newly excysted metacercariae has been described in

respect of Microphallus abortivus, Cercaria sevillana, M. primas etc. in which a

spiny tegument and circum oral sensory papillae are the features of common

occurrence (Saville and Irwin, 1991; Pina et al., 2007, 2011).

There are only a few reports on the occurrence of Microphallidae flukes in

vertebrates in India. So far only a few microphallid taxa have been described and they

include: Levinseniella indica, Basantisia ramai and Pseudospeloterma indicum from

birds (Lal, 1936; Pande, 1938; Murhar, 1960; Bharadwaj, 1962); Mehraformes

jabalpurensis and Microphallus indicus from reptiles (Bharadwaj, 1963; Mukherjee &

Ghosh, 1967); Megalatriotrema hispidum from the common frog (Rao, 1969); and

Spelotrema narii from intestine of jackal (Rao, 1965). In addition to these,

microphallid metacercarial stages have also been reported from sand crabs and

brackish-water prawns near the South eastern coasts of the Indian Sub-continent

(Anantaraman and Subramoniam, 1976; Jayasree et al., 2001). A comparison of the

morphological features of the various microphallid species described so far from

crustacean hosts in India reveals a close similarity of the present metacercarial stage

with Microphallus indicus Mukherjee & Ghosh, 1967 which was originally described

from a reptilian host. Yamaguti (1972) included M. indicus under a new genus

Allomicrophallus providing a new combination [Allomicrophallus indicus (Mukherjee

& Ghosh, 1967)], differentiating it from Microphallus on the basis of characters like

the intestinal caeca being better developed, long ejaculatory duct and the male papilla

being non protrudable. However, while reviewing the family Microphallidae, Deblock

(2008) considered Allomicrophallus Yamaguti, 1972 (with Microphallus indicus

Mukherjee & Ghosh, 1967 as its type species) and Bengaliniella Deblock, Mukherjee

& Ghosh, 1970 (with Microphallus dicaecus Mukherjee & Ghosh, 1967 as its type

species) synonyms of Megalatriotrema Rao, 1969. According Deblock et al. (1970),

the latter genus has already been considered a senior synonym of Bengaliniella and

Allomicrophallus because of the synonymy of the type species M. hispidum Rao, 1969

with M. dicaecus and with M. indicus. However, pending a restudy of the material of

Megalatriotrema hispidum, in our opinion the species Microphallus indicus should be

retained in the genus Microphallus as originally described by Mukherjee and Ghosh

31

(1967). In view of foregoing, the metacercarial type – III under the present study is

identified herewith as Microphallus indicus Mukherjee & Ghosh, 1967.

The pre-metacercarial larval stages of most digenean flukes undergo

development in snail, the first intermediate host; the morphological features of the

cercaria larva in most cases are group-specific (Smith, 1994). For example, members

of the family Paragonimidae characteristically have microcercous xiphidocercariae,

which typically have a spiny body surface, short stumpy tail, prominent penetration

glands and an oral stylet. Likewise, the fork-tailed or furcocercous cercariae are

typical of strigeid and schistosome flukes. On the basis of morphological features of

the tail and position of the ventral sucker, the cercarial types recovered from snails

were identifiable as distome leptocercous type and amphistome type. Since these

cercariae were not microcercous xiphidocercaria type, it may be assumed that

Paragonims infection does not commonly prevail in the study area and that the larval

stages recovered may be representing other trematode species instead.

The survey results suggest that while the mountainous crabs in colder, high

altitude foci of Arunachal Pradesh predominantly harbour metacercariae of

paragonimid flukes, P. westermani in particular, the same crustacean species in

relatively warmer localities of Meghalaya and Assam seemingly do not sustain the

infective stages of lungfluke. Instead, in these areas with a tropical climate, it is the

microphalid infections, and not the paragonimid, which emerged as the highly

prevalent infection. Further study in this direction is warranted so as to establish the

influence of climate variables on the epidemiology of Crustacea-borne parasitic

infections.

*****