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Parasitic
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JOURNAL OF PARASITIC DISEASES
(ISSN: 0971-7196)
Editor-in-Chief
Professor Prati Pal Singh
National Institute of Pharmaceutical
Education and Research
Sector-67, Phase-X
S. A. S. Nagar-160 062, India
Managing Editor
Prof. Varsha Gupta
Deptt. of Microbiology
Govt. Medical College & Hospital
Chandigarh-160 032
Assistant Managing Editors
Dr. Savita Singh
Mr. Sarbjit Singh Jhamb
National Institute of Pharmaceutical
Education and Research
S. A. S. Nagar-160 062
Advisory Board
Prof. R. C. Mahajan, Chandigarh
Dr. V. P. Sharma, New Delhi
Prof. M. S. Jairajpuri, Aligarh
Prof. N. K. Ganguly, New Delhi
Dr. G. P. Dutta, Lucknow
Editorial Board
Prof. J. Abdul-Salam, Kuwait
Prof. M. C. Agrawal, Jabalpur
Prof. H. S. Banyal, Shimla
Prof. Neelima Gupta, Bareilly
Prof. B. C. Harinath, Sevagram
Prof. C. J. Hiware, Aurangabad
Dr. S. L. Hoti, Pondicherry
Prof. P. D. Juyal, Ludhiana
Prof. C. Kalavati, Visakhapatnam
Dr. D. C. Kaushal, Lucknow
Prof. R. Madhubala, New Delhi
Dr. Pawan Malhotra, New Delhi
Prof. S. K. Malhotra, Allahabad
Prof. Nancy Malla, Chandigarh
Prof. S. C. Parija, Pondicherry
Dr. S. K. Puri, Lucknow
Dr. J. R. Rao, Izatnagar
Prof. Sarman Singh, New Delhi
Prof. Veer Singh, Sardarkrushinagar
Prof. M. L. Sood, Ludhiana
Dr. B. K. Tyagi, Madurai
Dr. Manita Williamson, Mumbai
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JOURNAL OF PARASITIC DISEASES
Volume 32, Number 1, June 2008
The official organ of
THE INDIAN SOCIETY FOR PARASITOLOGY
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JOURNAL OF PARASITIC DISEASES
Volume 32 Number 1 June 2008
CONTENTS
Invited review
Parasitic zoonotic infections in Egypt and India: an overview. Neelima Gupta,
D. K. Gupta and Said Shalaby
1-9
Review
Dot-ELISA in the diagnosis of parasitic diseases of animals. A. Gupta, A. K. Dixit
and Pooja Dixit
10-14
Original papers
Lymphatic filariasis in Benue State, Nigeria: community diagnosis using the rapidformat
antigen immunochromatographic card test. E. A. Omudu and F. C. Okafor
Purification and characterization of Plasmodium berghei glutathione reductase.
G. Kapoor, U. Bagai and H. S. Banyal
Human sparganosis in Sri Lanka: a case report and review of the cases.
R. L. Ihalamulla, H. Gunatilaka and S. D. Fernando
Immunocytochemical demonstration of 5-hydroxytryptamine and localization of
monoamine oxidase in Gigantocotyle explanatum and Gastrothylax crumenifer
(Digenea: Paramphistomidae). S. Ghani, Z. Ghani and S. M. A. Abidi
Seasonal dynamics in lungworm infections in sheep. N. Moghaddar,
S. S. Shekarforoush and A. Afrahi
Focus on pathogenic trematode cercariae infecting fresh water snails (Mollusca:
Gastrapoda) of tribal region of southern Rajasthan, India. S. L. Choubisa
On Paralueheia guptai n. gen. & n. sp. (Acanthocephala: Plagiorhynchinae Meyer,
1931) from Stromateus sinensis. A. M. Saxena and Rahul Gupta
Some epidemiological aspects of intestinal cestodes of sheep in a temperate climate.
K. A. Tariq, M. Z. Chishti and F. Ahmad
15-21
22-29
30-33
34-41
42-46
47-55
56-59
60-63

Short communications
Effect of the control of one-host cattle tick Boophilus microplus on growth and
haematological parameters of calves. S. Vatsya, R. R. Kumar, C. L. Yadav and R.
Garg
Mode of nutrition in pathogenic trematode larvae (redia and cercaria) which infect
hepatopancreas of fresh water snails (Mollusca: Gastropoda). S. L. Choubisa
Amoebic dysentery in dogs and dog owners. Saleh Umair, Azhar Maqbool,
Zubair Shabbir and M. D. Ahmad
Instructions to Authors
Undertaking by Authors
Membership Application
64-67
68-73
74-76
i-iv
v
vi

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 1-9
Invited review
J P D
Parasitic zoonotic infections in Egypt and India: an
overview
1 2 3
Neelima Gupta , D. K. Gupta and Said Shalaby
1
Department of Animal Science, M. J. P. Rohilkhand University, Bareilly.
2
Department of Zoology, Bareilly College, Bareilly.
3
Department of Complementary Medicine, Medical Division, National Research Centre, Cairo, Egypt.
Received 2 September 2008; revised 8 September 2008; accepted 17 September 2008
ABSTRACT. Zoonotic infections are diseases transmitted from vertebrate animals to man, and thus
compromise people's health as well as endanger their livelihoods by affecting livestock and/or other
useful domestic animals. Zoonotic diseases are an important group of infectious diseases within the
broader domain of veterinary public health. Changes in social, dietary and cultural mores and
environmental changes have caused a scourge in zoonotic diseases in Africa and Asia continents,
which World Health Organization has estimated to be the most zoonotic prone continents. Parasitic
zoonotic information in these continents are scattered, and the present communication attempts to
review the prevailing parasitic zoonotic diseases in these continents by selecting Egypt (Africa) and
India (Asia) as the two representative countries. Herein, the zoonotic infections in Egypt and India,
with reference to protozoal and helminthic diseases, are overviewed, which indicate the susceptibility
of the populations of these two countries to zoonotic diseases. The classification, mode of transmission
and control of zoonotic infections are also discussed.
Keywords: echinococcosis, filariasis, leishmaniasis, toxoplasmosis, zoonoses
INTRODUCTION
Zoonosis (Greek: “zoon” = animal) is an important
group of infectious diseases within the broader domain
of veterinary public health. Food and Agricultural
Organization/World Health Organization (WHO)
expert committees call it “the infectious diseases that
are naturally transmitted between animals and man”.
The diseases transmitted from humans to animals are
sometimes called reverse zoonoses or anthroponoses
(Greek: “anthropos” = man, “nosos” = disease, Noble
and Noble, 1982). During the last 20 years, 70% of the
emerging diseases have been found to be zoonotic in
Corresponding author: Prof. Neelima Gupta, Department of
Animal Science, M. J. P. Rohilkhand University, Bareilly-243
006, India. E-mail:guptagrawal@rediffmail.com
nature, and about 300 diseases are common to both man
and animals. Human health is inextricably linked to
animal health and production. The close association
between animals and man can lead to serious risk to
public health with severe economic consequences.
Consequently, a number of communicable diseases
(zoonoses) can be transmitted from animals to humans.
Many of them have been reported infrequently or
locally only, whereas some are relatively unimportant
from both public health and economic points of view.
Zoonoses affect man in a variety of ways by causing
illness, delays, termination, monetary losses in the form
of cost of drugs and physician's fee, loss of wages and
work efficiency, medico-legal complications and
unfavorable publicity.
A zoonotic agent may be a bacterium, a virus, a fungus

2 Gupta et al.
or a parasite like protozoan or helminth. Some of these 7. Fish and shell fish: Heterophyes
pathogens cause diseases in humans but sub-clinical
heterophyes, Diphyllobothrium latum,
infection in animals makes it difficult to eradicate the
Capillaria phillippinesis, Clonorchis
diseases caused by them. Over 75% of the new diseases
sinensis, Gnathostoma spinigernum,
that have affected humans over the past 10 years have
Angiostrongylus cantonensis (eating
been caused by pathogens originating from an animal or
infected snails or crabs), Prohemistomum
products of animal origin. Many of these diseases have
vivax and Haplorchis pumilio.
the potential to spread through various means over long
distances to become global problems. A vast majority of
Zoonoses has also been classified according to the
them are not prioritized by health systems at national
ecosystem in which they circulate (Hubalek, 2003):
and international levels and are, therefore, labeled as 1. Synanthropic zoonoses: Urban (domestic)
neglected diseases.
cycle in which the source of infection is
The emerging interdisciplinary field of conservation
domestic and synathropic animals
medicine, which integrates human and veterinary 2. Exoanthropic zoonoses: Sylvatic (feral and
medicine as well as environmental sciences, is largely
wild) cycle a natural focci outside human
concerned with zoonoses. The overall concept of
habitats.
zoonoses is complex. It involves man, an invertebrate
(usually arthropod), the causative agent and the 3. Both urban and natural cycles.
environment – all forming a biological whole (Shah, 1987).
MODES OF TRANSMISSION
Zoonotic diseases in Africa and Asia are of importance
Zoonotic diseases may be transmitted by one of the
as these continents are especially prone to parasitic
following modes (Shalaby and Gupta, 1997)
zoonotic diseases. Keeping this in view, the present
study was undertaken to compare the zoonotic diseases 1. Inhalation
prevalent in an African country, Egypt and an Asian
country, India.
2. Ingestion: Via meat e. g. beaf as in Taenia
saginata, pork as T. solium, trichonosis
CLASSIFICATION OF ZOONOSES
and Balantidium coli.
Zoonoses have been classified according to their 3. Contact with animals
reservoir hosts:
4. Arthropod vectors
1. Cattle: Taenia saginata, screw worm,
Babesia bovis and toxoplasmosis.
2. Sheep and goat: Fasciola hepatica.
3. Pigs: Balantidium coli, T. solium,
Trichenella spiralis, Fasciolopsis buski
and isosporiasis.
4. D o g s : D i p y l i d i u m c a n i n u m ,
Echinococcus granulosus, Ancylostoma
caninum, toxocariasis, reservoir for
Leishmania donovani, L. tropica and
Chagas disease,
•Fleas as Diphylidium caninum,
Hymenolepis nana, H. diminuta.
•Mosquito-borne infections
•Tick-borne disease such as
babesiosis.
•Sandfly-borne such as leishmaniasis.
PARASITIC ZOONOTIC DISEASES
Protozoal zoonoses
5. Cats: Toxocara cati, toxoplasmosis and as Leishmaniasis: Three varieties of leishmaniasis have
reservoir for leishmaniasis and Chagas been reported; cutaneous, mucocutaneous (Espundia)
disease.
and visceral (Kala Azar, Dum dum fever or ponos),
which are serious, debilitating and disfiguring
6. Rodents and rats: Hymenolepis nana, H. diseases. Cutaneous leishmaniasis (CL) is also known
diminuta.
as 'bouton d'Orient, 'Oriental sore', 'Baghdad boil',
Delhi sore and 'Aleppo button'. The causative agents are

Parasitic zoonotic infections in Egypt and India
3
generally Leishmania tropica, L. major and L. Toxoplasmosis: Toxoplasmosis is caused by
aethtiopica. The infection produces a skin ulcer which Toxoplasma gondii that is known as a potential parasite
leaves an unsightly scar on healing.
for man for many years but its true nature as a coccidian
was discovered during 1969-70 (Smyth, 1994).
Leishmaniasis of man are generally transmitted by the
Domestic cats predominate as reservoir at home and in
bite of female sandflies. However, the distribution and
the laboratory environment. Mice, rats, hamsters,
transmission is complicated by the fact that the disease
guinea pigs, rodents, rabbits, dogs, sheep and cattle are
is a zoonosis and there are a wide range of reservoir
intermediate hosts. Man acquires infection either by
hosts. CL is associated with wild rodents and dogs,
direct ingestion of oocysts from a cat or by eating raw or
which facilitate transmission to humans and cause
undercooked meat infected with tissue cysts of
zoonosis. The known reservoir hosts have been
Toxoplasma sp. Cooks or butchers or persons handling
reviewed by WHO (1984).
raw meat are particularly at risk. Any cat, no matter how
From Egypt, Morsy et al. (1995) reported that zoonotic well cared for, may be passing Toxoplasma oocysts. As
CL was the most important zoonotic disease in the the disease is congenital, therefore, a pregnant woman
country. The results showed that rodent population is is advised not to handle cat's faeces.
more or less common in Al-Arish City (Rattus rattus
In Egypt, the disease is responsible for a significant
and R. norvegicus ), Bir Lehfan (Gerbillus pyramidum)
percentage of abortions among inhabitants of Egyptian
and Abo Oegela ( G. pyramidum , Meriones crassus and
cities and rural areas . Babies may have problems of
Jaculus jaculus). Phlebotomus papatassi is the main
vision after being delivered normal at a later age
vector being common in nearly the whole of Sinai.
(Shalaby,1989; Abdelhamed, 2008) .
Isolates recovered from P. papatassi trapped in Sad Al-
Rawafei and G. pyramidinum trapped in Abo Ogela From India, Chhabra et al. (1981) discussed the
proved (isoenzyme cellulose acetate electophoresis) to epidemiological aspects and public health potential of
be L. major biochemically indistinguishable from each subclinical toxoplasmosis in dairy goats with reference
other. .
to consumption of undercooked goat's meat and raw
goat's milk. Toxoplasmosis in Indian AIDS patients has
From India, Sharma et al. (1973) studied the
been recognised as an opportunistic infection from
epidemiological and entomological features of an
India. Mittal et al. (2005) recorded the prevalence of
outbreak of CL (caused by L. tropica) in Bikaner city,
toxoplasmosis in Indian women of child bearing age,
Rajasthan. Infection with L. major has been reported to
which was due to ingestion of undercooked or
be a typical zoonoses in the dry terrain of the Rajasthan
uncooked meat. Sundar et al. (2007) evaluated the
canal region, whereas in Bihar, most of the cases of
seroprevelence of T. gondii in healthy adult population
Kala Azar were caused by L. donovani (Peters et al.,
of blood donors in Karnataka.
1981). Mittal et al. (1991) evaluated the sensitivity and
specificity of three serological tests, indirect Giardiasis: Animals considered to be sources of
immunoflourescent antibody (IFA) test, enzyme- infection include rodents (rats, voles, beavers,
linked immunosorbant assay (ELISA) and counter muskrats), dogs and cats and hoofed animals, beavers
immunoelectrophoresis (CIEP) for the diagnosis of appear to be the animals most commonly infected
Indian Kala Azar. The zoonotic reservoir of Kala Azar (Bemrick and Erlandsen, 1988). Dogs are implicated as
has been identified in Bihar, where more than 1,00,000 zoonotic source of infection (Fayer et al., 2004) for
cases alone were reported out of 4,00,000 cases giardiasis. Although the majority of patients with
recorded worldwide (Kishore and Kar, 1996). Singh chronic diarrhoea in Egypt suffer from Entamoeba
(2006) estimated more than 90% of visceral histolytica infection; nevertheless, there are some cases
leishmaniasis (VL) to be from India, Bangladesh, of Giardia lamblia infection being mostly of zoonotic
Southern Sudan, Nepal and north east Brazil. He origin.
suggested various modes of parasite transmission
including vector-borne, blood transfusion, needle
Malaria: Malaria is one of the world's greatest killers.
sharing and sexual (person to person). Sundar et al.
One of the surprising research developments has been
(2007) conducted a randomized, controlled, phase 3
the discovery that certain species of simian malaria are
open label study comparing paromomycin with
capable of infecting man and thus causing zoonosis.
amphotericin B for the standard care and control of
The blood forms of Plasmodium knowlesi are known to
Kala Azar in Bihar, India.
be transmissible to man, though it was earlier believed

4
Gupta et al.
that monkey malaria was not usually infective to man. reported. Filariasis caused by W. bancrofti is most
During the 1960's, the concept changed and the report common. The disease causes lymphatic obstruction
that malaria from apes and monkeys can be transmitted with swollen legs or in some cases, even the whole
to humans and the first proof of a simian malaria lower half of the body may be swollen . The morbid
parasite causing zoonosis in man was reported by Chin changes initiated by the parasite are initially confined to
et al. (1965), which involved a case of natural infection the lymphatic system and the clinical manifestations are
of man by P. knowlesi, a species normally known to commonly called wuchereriasis (Filariasis). Filariae
infect monkeys. Although the prevalence of malaria is occur in a wide range of tissue habitats: lymph glands,
high but zoonotic cases are comparative few. From deep connective tissues, subcutaneous tissues or
India, Das et al. (1996) recorded zoonoses by mesenteries. The revolting fleshy deformities are
sporozoans from West Bengal, where zoonotic malaria commonly termed elephantiasis.
infection was reported in a 23 year old student who
inhabited an area of gibbons which carry a malarial
Strongyloidiasis: Many vertebrates, not only
parasite similar to Plasmodium.
mammals but also amphibians, reptiles and birds are
affected by th strongyloidiasis. The available source of
Sarcocystosis (Sarcosporidiosis): Sarcocystosis, the laboratory material is the faeces of farm animals
obligatory heteroxenous parasite produces muscle containing eggs and larvae of the parasite. Best known
cysts in intermediate hosts such as domestic herbivores species are Strongyloides stercoralis in man, cat and
(cattle, sheep, pigs) and rodents. Final hosts are dog; S. fulleborni in primates other than man; S.
predators such as dogs, cats, owls, snakes and man. papillosus in ruminants; S. ratti in rodents; S. ransomi
Cases of human infection in muscles have been in pigs and S. westerni in equines. In man, S. stercoralis
reported, probably from organisms normally found in causes strongyloidiasis, a major intestinal infection
monkeys (Beaver et al., 1979).
(Grove, 1989), which is widely distributed in the tropics
and subtropics. The pathology of the disease has been
Work on prevalence, morphology, life cycle,
reviewed by Georgi (1982) and Grove (1989).
transmission, pathogenesis immunology, biochemistry
Strongyloidiasis, caused by S. stercoralis, is always
and prophylaxis of sarcocystosis in domestic animals of
found as a natural infection in dogs and cats in Egypt
India was done by Juyal and Cross (1991), who
and few cases of zoonosis in man have been reported.
correlated the prevalence of sarcocystosis with foodborne
parasitic zoonosis and stressed upon the Angiostrongylosis: The disease is caused by the
emerging problems in food-borne parasitic zoonosis nematode worm Angiostrongylus that is normally
and their impact on agriculture and public health. found in dogs and the intermediate hosts are land snails
Helminthic zoonoses
and slugs. Canine angiostrongylosis is usually a chronic
condition extending over months or even years.
A. Nematodes
Sometimes the infection is zoonotic in humans. The
parasite can cause the syndrome of meningoen-
Trichinoses (Trichinellosis): Trichinoses is due to cephalitis. The first record of the disease to be zoonotic
Trichinella spiralis infection where the reservoirs are was from Formasa (Chatterjee, 1995). Human
swine, dogs, cats and rats. It is a serious and often fatal infection occurs by ingestion of raw vegetables
disease. This is essentially a 'domestic' or 'synanthropic' containing the third stage larva of the parasite or
parasite and has since long been recognized to cause ingestion of tissues of improperly cooked infected
zoonosis and is transmitted to man by the ingestion of intermediate hosts (snails) and carrier or paratenic host
infected pork or sausages. The disease often remains (crabs, prawns, pigs). Infection may also occur by
undetected during life and is thus an important health drinking contaminated water containing the infective
problem.
larvae. The global dispersal of peridomestic zoonoses,
angiostrongylosis caused by A. cantonensis, the
Filariasis: The disease is caused by the filarial worm,
lungworm of rats and eosinophilic meningitis in
Wuchereria bancrofti which is a widely distributed
humans due to the worm has also been reported (Kliks
parasite. Filarial parasites are probably the most
and Palumbo, 1992; Mahajan et al., 1992).
important group of helminthic infection in humans but
they are of marginal concern to the veterinarian since Ascariasis: Ascaris lumbricoides, a parasite of man
domestic animals are of little significance in their and pigs is cosmopolitan in distribution. The infection
epidemology and few cases of zoonotic infections are rate of Ascaris is high and upto 90% of the population

Parasitic zoonotic infections in Egypt and India
5
may suffer from ascariasis. The infection results in the first record of heterophyid infection was made by
malnutrition and retardation in growth of children, Mahanta et al. (1995) from Dibrugarh, Assam. Only
pneumonitis, asthma, diarrhoea, nausea, abdominal two cases were reported to have passed eggs in their
pain and anorexia. In some cases, the infection may stools and due to the paucity of availability of these
become zoonotic. Ascariasis and visceral larval parasites from India, their pathogenecity is practically
migrants are always found in rural Egypt or even in untouched.
urban primary school children who are fond of playing
with dogs.
Black Spot Disease: The disease is caused by the
metacercaria of trematodes. From India, the disease has
B. Trematodes been reported in Catla catla, Cirrhina mrigala, Labeo
rohita and Schizothorax richardsonii due to
Fascioliasis: Fascioliasis commonly occurs in sheep,
metacercarial infection of Neascus and Diplostomum
goats and cattle although a wide range of other hosts
species (Malhotra and Bannerjee, 1989). The black
have been reported. Man is usually not considered to be
spots represent encysted metacercaria surrounded by
a host of Fasciola hepatica but the eating of water cress
th
reaction pigmentation and outer cysts of 1/200–1/7 of
appears to be a common source of human infection
an inch in diameter surround the parasite cysts, the cysts
(Marsden and Warren, 1984). Although the usual site
thus consist of an outer thick cellular layer (of host
of infection is the liver but in aberrant hosts like man,
origin) and an inner thin non-cellular layer (of parasite
the lung may be involved. Human fascioliasis infection
origin). The intense black colour of the cysts is attained
occurs after swallowing encysted matacercariae on
due to the presence of melanocytes in the outer layer.
unwashed green lettuce. The irrigation of this plant by
Not only can the older cysts rupture to enable
contaminated water with cercaria from water drains
reinfection/fresh infection in other fish, but the
containing the intermediate host snail and exposed to
metacercaria may also be directly transmitted by
faeces of infected cattle and sheep helps in propagation
drinking of infected water, and consumption of such
of infection (Beaver et al.,1984; Shalaby and Gupta,
uncooked infected fish can cause infection in man. The
2000).
parasite can be directly transmitted during extensive
Schistosomiasis: Schistosomes are the blood flukes of parasitaemia (Bannerjee and Malhotra, 1989) and can
mammals and birds. It commonly lives in the veins of also be zoonotically transmitted (Neascus vetesai and
the mesenteries of the colon of man, wild rodents and N. channi) to homeotherms (Malhotra and Chatterjee,
primates, and causes a debilitating disease commonly 1996). The zoonotic potential and environmental
known as schistosomiasis. Schistosomiasis is perhaps interactions in fish parasite systems was discussed by
the most important disease of helminthic origin in the Malhotra and Chatterjee (1996) and the dogma of black
world today. The disease is caused by the trematode spot disease in Garhwal Himalayas and freshwater
parasite Schistosoma, which parasitises all domestic ecosystems of gangetic plains was also discussed
mammals mainly sheep and cattle. Water snails are (Malhotra et al., 2002) but its zoonotic signifance has
important in the transmission of the disease. From not been pointed out by the authors.
India, the possibility of schistosomiasis in man by S.
incognitum (normal host Macaca mulatta) has been
C. Cestodes
discussed by Ahluwalia (1972).
Echinococcosis (Hydatid disease): Echinococcus
granulosus, commonly called as the hydatid worm,
Heterophyiasis: Heterophyiasis infection caused by
parasitises members of the canine family and foxes as
Heterophyes heterophyes, an intestinal parasite of dogs,
adults but humans can aquire zoonotic infection with
cats, fox, humans is commonly found in Asia and Egypt
hydatid larvae. E. granulosus causes one of the most
and has been introduced into Hawaii. Reports also exist
serious larval tapeworm infections in man. The larva of
from Palestine and Far East. Eating of raw or uncooked
this very dangerous species is the cause of alveolar
fish causes heterophyiasis in humans. The disease is not
(multilocular) hydatid diseases in man, has variable
common in India. Gill (1972) recorded the parasite in
clinical manifestations making diagnosis complex and
two out of 88 cats sampled at All India Institute of
difficult. Normal hosts for the adult parasite are dogs in
Medical Sciences, New Delhi, although helminth
which hundreds of worms may occur in small intestine.
parasites were present in 84 samples. The first record of
Wolves and jackals also harbour the adult worm and in
H. dispar from Madras at an infection rate of 96% was
some areas, foxes are probably affected. Human
reported by Rajavelu and Raja (1988). From humans,
echinococcosis (hydatidosis) is commonly the result of

6
Gupta et al.
intimate contact with dogs; however, other unique cases of zoonosis by mites, O. bacoti (usual hosts rats)
ethnic customs favour infection. Animals primarily in two laboratory personnel and six veterinary students
involved are dogs, sheep, cattle, swine, rodents and in Haryana were recorded by Ram et al. (1986).
deer. Man becomes infected by eating faecal eggs,
contaminated water or food stuffs or from handling
CONTROL
infected dogs especially on farms resulting in the Intervention and control of zoonoses requires concerted
ingestion of eggs.
efforts between the veterinary and human health sectors
Echinococcosis in Egypt causes cystic disease in liver,
as it requires both men and animals. WHO has taken the
lungs and long bones. The definitive host is carnivorous
lead in bringing together international and national
animals, especially dogs. The eggs are passed in dog's
organisations to deal with the problems posed by both
faeces and are accidentally swallowed by humans.
emerging and endemic zoonoses. The control methods
From India, Parija (1991) employed serodiagnostic
may target:
tests to detect circulating hydatid antigen (Cag) in the
serum by ELISA and other assays in humans.
1. The human host (treatment, immunization).
2. Insect, tick or mite vectors (repellents, insect
Hymenolepiasis: Hymenolepiasis caused by sterilization, insect traps, insecticidal sprays,
Hymenolepis diminuta is also frequently seen in rural
bed nets, naturalistic methods eg. drainage,
areas of Egypt where bread, cereals or any stored food is
vegetation clearing, biological control
left uncovered, being exposed to reservoirs of this
methods using bacteria or viruses pathogenic
worm, rodents, fleas, beetles and cockroaches. Usually,
for the vector).
swallowing of the infected arthropod causes this
infection. The definitive hosts of the parasites are rats 3. Animal reservoir (treatment, elimination,
and other rodents and are occasionally found in other
immunization).
mammals including man.
4. Deep freezing (-15°C for 20 days before
Dipylidiasis: Dipylidiasis is caused by the cestode
distribution of meat).
parasite, Dipylidium caninum. It is the commonest
tapeworm genus of domestic dog and cat and rare cases
Parasitic zoonotic infections: Egypt and India
of human zoonoses have been reported. Children are Egypt
probably infected by having their faces licked by a dog
just after the dog has nipped a flea, as the fleas are the In Egypt, studies on parasites transmitted to man
intermediate host of the parasite. The infection may also through fish have been conducted by Shalaby and his
result by accidental ingestion of infected fleas while collegues since 1980 till date. Many parasites of
fondling cats and dogs.
zoonotic importance have been reported: Aliptrema
tilapia was recorded as a new parasite of zoonotic
From India, a case in a child from Calcutta has been importance occurring by eating improperly cooked
reported from which two adult worms were recovered Nile fishes (Shalaby, 1993). Phagicola ornamentata
(Chatterjee, 1995). A unique case of dipylidiasis and Pharyngostomum flapi were recorded as new
infection with D. canimum in a four year old boy is said species (Shalaby Esposito et al., 1993). On the other
to have been acquired through pet cats as a zoonotic hand, Prosostephanus industrius, Procerovum
infection (Gadre et al., 1993).
calderoni and Moedlingeria amphoraeformis were
MITES
recorded for the first time from Egypt (Shalaby et al.,
1993). Phagicola ornamentata and Pharyngeostomum
Ornithonyssus bacoti is the tropical rat mite. It is mostly flapi were reported as new species resulting due to
found on rats but also attacks mites and even man and infection from Tilapia nilotica, whereas P. calderoni
may give a painful bite. It also acts as a vector of and M. amphoraeformis were again recorded from
Litomosoides carinii, a filarial nematode parasitic in Egypt for the first time. Lake Quarun (Fayoum
rodents. They serve in the transmission of certain province) fishes were investigated where Mugil
rickettsial and viral diseases. The mite plays a minor cephalus and M. capito were found infested with both
role in transmission among reservoir hosts and Heterophyidae and Diplostomatidae encysted
occassionally to man of endemic typhus, rickettsial metacercariae in their muscles. Solea vulgaris showed
pox, Q fever, tularimia, plague and viruses. Sporadic diplostomidae encysted metacercariae in their muscles.

Parasitic zoonotic infections in Egypt and India
7
Prohemistominae encysted metacercariae were again Twenty-one contributions were presented on
encountered for the first time from M. cephalus. Worms “Zoonoses” in the Symposium of Indian Association
caused acute enteritis in case of heavy infestation for Advancement of Veterinary Parasitology in 1987,
(Shalaby et al., 1996). Shalaby et al. (2002) reported and 24 papers were presented in a Seminar on Zoonoses
that human fish-borne trematode helminthoses caused in 1989, which discussed the diseases transmissible
gastrointestinal symptoms such as indigestion, from animals to man including protozoan and helminth
diarrhoea and abdominal pain including heart problems diseases. Dutta (1991) and Dutta and Dutta (1991)
ranging from arrhythmia to ventricular premature beats presented a background information on zoonoses in a
as well. Sometimes obstructive jaundice, complicated seminar on Zoonoses. The Global Meet on Parasitic
by sepsis and disseminated intravascular Diseases in 1996 also focussed on Zoonoses in a
coagulopathy–like syndrome was evident. Shalaby et Symposium. Zoonoses maybe said to consist of five
al. (2006) reported Heterophyes heterophyes, H. h. components: parasites, maintenance/reservoir host,
nocens, H. katsuradai , H. dispar and H. aequalis as intermediate host and vector, susceptible/potential host
fish-borne helminth zoonoses and stated that diarrhoea and environment, and each of these components may
was the main clinical manifestation. interact with the other (Fig. 1).
India
Zoonotic diseases have been a favoured topic for
organising symposia in India. During the Asian
Congress of Parasitology held at Bombay in 1978,
zoonoses occupied an important place for deliberations.
Food-borne parasitic zoonoses have a major impact on
the health and economy in developing countries in the
tropics and sub-tropics. Throughout India, problems of
food-borne parasitic zoonoses differ due to varied food
habits. Bhatia (1991, 1992) reviewed the status of
various food-borne parasitic diseases in India and put
Intermediate
host and vector
Susceptible/
potential host
ZOONOSES
Maintenance/
Reservoir host
Environment
Parasites
Fig. 1. Components of zoonoses and their interaction

8
Gupta et al.
forth its impact on the health and economy in
developing countries and observed variations in foodborne
parasitic zoonoses due to varied food habits,
unhygienic living conditions, lack of education, poor
personal hygiene, poverty and occupation.
The comparative reports on zoonoses in Egypt and
India presented herein bring forth some of the important
parasitic zoonotic diseases of the two countries, which
report information on the incidence, occurrence,
clinical manifestations and diagnostic aids. It is
envisaged that future studies will stimulate scientists
for further research in this area to establish parasitic
zoonotic disease potential for each disease.
REFERENCES
Abdelhamed AH. 2008. Studies on Toxoplasma gondi of animal
origin in relation to infertility and abortion in pregnant 22:138-141.
women. M.V. Sc. (Zoonoses), Cairo University.
Fayer R, Dubey J and Lindsay D. 2004. Zoonotic protozoa from
land to sea. Trends Parasitol 20: 531-536.
Gadre DV, Kumar A and Mathur M. 1993. Infection by
Diphlidium caninum through pet cats. Ind J Ped 60:151-152.
Georgi JR. 1982. Strongloidiasis. In: CRC Handbook Series in
Zoonoses. Section C: Parasitic Zoonoses, Vol. II. M. G.
Schultz (Ed.), pp. 257-267.
Gill HS. 1972. A study of the parasitic zoonoses in India. Proc 59
Ind Sci Cong Assoc. 549.
Grove D I. 1989. Strongyloidiasis. A major roundworm infection
of man. Taylor and Francis, London.
Hubalek Z. 2003. Emerging human infectious diseases:
Anthroponoses, Zoonoses and Sapronoses. Emerg Infect Dis
9:403-403.
Juyal PD and Cross JH. 1991. Sarcocystis and Sarcocystosis in
India. Southeast Asian J Trop Med Public Health Suppl
Kishor K and Kar SK. 1996. Zoonotic reservoir of Kala Azar.
th
Ahluwalia SS. 1972. Zoonotic potentials of Schistosoma Proc 8 Nat Cong Vet Parasitol Nat Symp Mol Parasitol 30-
incognitum. Indian J Anim Sci 42:962-964. 31.
th
Bannerjee S and Malhotra SK. 1989. Biology and pathogenic
potential of black spot trematodes in high altitude fishes of
India. Curr Sci 58:498-500.
Beaver P, Jug R and Gupp E. 1984. Clinical Parasitology, 9 th.
Edn. Philadelphia: Lea and Febiger.
Beaver PC, Gadgil R K and Morera P. 1979. Sarcocystis in man: a
review and report of five cases. Amer J Trop Med Hyg
28:819-855.
Bemrick WJ and Erlandsen SL. 1988. Giardiasis – is it really a
zoonosis? Parasitol Today 4:69-71.
Bhatia BB. 1991. Current status of food-borne parasitic zoonoses
in India. Southeast Asian J Trop Med Public Health Suppl.
22:36-41.
Bhatia BB. 1992. Food –borne parasitic zoonoses in India.
Pashudhan 7:1-5.
Chatterjee, K D.1995. Parasitology. Chatterjee Medical
publishers, Calcutta. 238 pp.
Chhabra MB, Bhardwaj RM, Gautam OP and Gupta RP. 1981.
Toxoplasma infection and abortion in dairy goats. Trop Anim
Health Prod 13:222-226.
Chin W, Contacos P G, Coatney G R and Kimball H R. 1965. A
naturally acquired quotidian type malaria in man transferable
to monkeys. Science 149:865.
Das S, Malakar P, Saha GK, Dasgupta B and Hati AK. 1996. A
case of Plasmodium malariae infection in the Dooars region
of West Bengal, India. Indian J Malariol 33:159-160.
Dutta LP. 1991. Background information on zoonoses. Proc Sem
Zoonoses 138.
Dutta LP and Dutta LP. 1991. Background information on
zoonoses. Proc Sem Zoonoses 123-128.
Kliks MM and Palumbo NE. 1992. Eosinophilic meningitis
beyond the Pacific basin: the global dispersal of a
peridomestic zoonoses caused by Angiotrongylus
cantonensis, the nematode lungworm of rats. Soc Sci Med 34:
199-212.
Mahajan RK, Almeida AJ, Sengupta SR and Renapurkar DM.
1992. Seasonal intensity of Angiostrongylus cantonensis in
the intermediate host, Laevicaulis alle. Intl J Parasitol 22:
669-671.
Mahanta J, Narain K and Srivastava VK. 1995. Heterophyid eggs
in human stool samples in Assam: First report from India. J
Comm Dis 27:142-145.
Malhotra SK and Bannerjee S. 1989. A review of the
environmental correlates and systematics of the organisms of
black spot disease in Indian fishes. In: Dr. B.S. Chauhan
Comm. Vol. 87-101.
Malhotra SK and Chatterjee S. 1996. Zoonotic potential and
environmental interaction in fish parasite systems. I.
Zoonoses and seasonality of black spot disease. In: Parasitic
Diseases- New Horizons. Banerjee DP, Ghosh JD and Gupta
SK (Eds.) 121-128.
Malhotra SK, Geetanjali and Chatterjee S. 2002. The dogma of
black spot disease in Garhwal Himalayas and freshwater
ecosystems of gangetic plains – will the fish managers wake
up? Univ Alld Stud (New Millen. Ser.) 1:45-56.
Marsden PD and Warren KS. 1984. Fascioliasis. In: Tropical and
Geographical Medicine. Warren KS and Mahmoud AAF
(Eds.), pp. 458-460. McGraw Hill, New York.
Mittal V, Bhatia R and Sehgal S. 1991. Serodiagnosis of Indian
Kala Azar. Evaluation of IFA, ELISA and CIEP tests. J Comm
Dis 23:131-134.
Mittal V, Bhatia R, Singh VK and Sehgal S. 2005. Prevalence of

Parasitic zoonotic infections in Egypt and India
9
toxoplasmosis in Indian women of child bearing age. Ind J Shalaby SI, Easa M, El S and Afify MH. 1996. Lake Quarun
Pathol Microbiol 38:143-145.
fishes as intermediate hosts for transmitting some
trematodes. Egypt J Comp Pathol and Clinical Pathol 9: 201-
Morsy T, Naser A, El-Gibali M, Anwar A and El-Said A. 1995.
213.
Studies on zoonotic cutaneous leishmaniasis among group of
temporary workers in North Sinai Governorate, Egypt. J Shalaby SI, Esposito P, Riegler G, Di- Carlo V and Carratu R.
Egypt Soc Parasitol 25:99-106.
1993. Trematode parasites transmitted to man and fish-eating
mammals through Tilapia nilotica. II-New trematode species
Noble ER and Noble GA. 1982. Parasitology. The biology of
. Acta Mediterranea Di Patologia Infective Tropicale12:111-
animal parasites. Leas and Febiger, Philadelphia. 522 pp.
114.
Parija SC. 1991. Recent trends in serodiagnosis of hydatid
disease. Southeast Asian J Trop Med Public Health Suppl
22:371-376.
Peters W, Chance ML, Chowdhury AB, Dastivar BG, Nandy A,
Kalras JL, Sanyal RK, Sharma MID, Srivastava I and Schnur
LF. 1981. The identity of some stocks of Leishmania isolated
in India. Annals Trop Med Parasitol 75: 247-249.
Rajavelu G and Raja EE. 1988. On helminth parasites in
domestic cat in Madras. Cheiron 17:11-14.
Shalaby SI, Mohamed I and Neelima Gupta. 2002. Clinical and
parasitological studies on human fish-borne trematode
helminthoses with emphasis on intestinal histoenzymatic
changes in Egypt; comparative records from India. Ind J
Environ Ecoplan 6:409 -414.
Shalaby SI, Riegler G, Esposito P, Russo MI and Carratu R.
1993. Trematode parasites transmitted to man and fish-eating
mammals through Tilapia nilotica. I- Experimental infection.
Acta Mediterranea Di Patologia Infective Tropicale 12:107-
110.
Ram SMT, Satija KC and Kaushik RK. 1986. Ornithonyssus
bacoti infestation in laboratory personnel and veterinary Sharma MID, Suri JC, Kalra NL, Mohan K and Swami PN. 1973.
students. Intl J Zoonoses 13:138-140.
Epidemiological and entomological features of an outbreak
of cutaneous leishmaniasis in Bikaner during 1971. J Comm
Shah HL. 1987. An integrated approach to the study of zoonoses. Dis 5:54-72.
J Vet Parasitol 1:1-2.
Singh S. 2006. New development in diagnosis of leishmaniasis.
Shalaby SI. 1989. On zoonotic toxoplasmosis. Egypt J Comp Ind J Med Res 123:311-330.
Pathol and Clinical Pathol 2:225-254.
Smyth JD. 1994. Introduction to Animal Parasitology.
Shalaby SI. 1993. Communicable parasites from Nile fishes. I-
Pathological and parasitological studies on Aliptrema tilapi
n.sp. Egypt J Comp Pathol and Clinical Pathol 6:199-2005.
Shalaby SI and Gupta Neelima. 1997. Zoonotic diseases with
emphasis on parasitic infestations. Him J Env Zool 11:157- Res 126:50-55.
162.
Cambridge University Press, Cambridge. pp.549.
Sundar P, Mahadevan A, Jayshree RS, Subbakrishna DK and
Shankar SK. 2007. Toxoplasma seroprevalence in healthy
voluntary blood donors from urban Karnataka. Ind J Med
Sundar S, Jha TK, Thakur CP, Sinha PK, Bhattacharya SK. 2007.
Shalaby SI and Gupta Neelima. 2000. Clinical studies on Injectable paronomycin for visceral leishmaniasis in India.
zoonotic diseases in Major Cairo (Egypt) and a review on New Eng J Med 356: 2571-2581.
zoonotic potential in India. J Parasit Appl Anim Biol 9:17 -
24 .
WHO. 1984. The leishmaniasis. Report of a WHO expert
committee. Technical Report series no. 701. pp. 140.
Shalaby SI, Anter O, Hassan N, El- Mahdy M, Mohamed I and
Gupta Neelima. 2006. Clinical manifestations of
experimentally induced fish-borne trematode infection in
experimental animals. J Curr Sci 9:443-448.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 10-14
Review
J P D
Dot-ELISA in the diagnosis of parasitic diseases of
animals
1 1 2
A. Gupta , A. K. Dixit and Pooja Dixit
1
Department of Parasitology, College of Vetrinary Science and Animal Husbandry, Jabalpur.
2
Department of Clinical Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Rewa.
Received 18 August 2008; accepted 24 September 2008
ABSTRACT. Traditional serological tests such as indirect haemagglutination, complement fixation,
counter immunoelectrophoresis and immunofluorescence are tedious and difficult to standardize,
conduct and interpret. In the last decade, enzyme-linked immunosorbent assay (ELISA) has been
developed to detect a large number of parasitic diseases. However, this assay also has a number of
drawbacks. In an effort to develop a relatively more simple to perform and inexpensive to use assay,
ELISA was modified to Dot-ELISA. The Dot-ELISA has been widely accepted as a rapid and versatile
assay for the detection of protozoan and metazoan diseases in humans and livestock.
Keywords: babesiosis, Dot-ELISA, fasciolosis, haemonchosis, serodiagnosis
INTRODUCTION
The Dot-ELISA for the demonstrartion of the parasite
specific antibodies depends upon the principle that
when the test serum is layered on the nitrocellulose
membrane bound with the parasitic antigen, the specific
antibodies if present in the serum, will bind to the
corresponding antigen dot. The antigen detection Dot-
ELISA may be conducted in two ways. Specimens
Corresponding author: Dr. A. K. Dixit, Department of
Parasitology, College of Vetrinary Science And Animal
Husbandry, Jabalpur-482 001, India.
E-mail: alokdixit7@ yahoo.com
The Dot-enzyme-linked immunosorbent assay (Dot-
ELISA) is the most frequently used assay to
demonstrate either serum antibodies or antigens in the
diagnosis of parasitic diseases. The Dot-ELISA has
become increasingly popular as a field assay for the
diagnosis of many of the parasitic diseases including
fascioliasis, toxocarosis, dirofiloriosis, haemonchosis,
trichinellosis, leishmaniosis and babesiosis.
suspected of containing parasite antigens can be dotted
in minute volumes directly on to the nitrocellulose (NC)
paper and allowed to dry. Alternatively, antigen in test
samples may be captured using a sandwich Dot-ELISA
that is capture anti body (monoclonal or polyclonal) is
dotted on to the filter paper. The test sample is then
incubated with antibody filter discs which specifically
capture parasite antigen. After washing to remove
unbound antigen and extraneous material, a second
antibody specific for antigen is added, effecting a
sandwich. After incubation with antigen specific
antibody and/ or enzyme conjugated anti-antibody, the
addition of a precipitable, chromogenic substrate
causes the formation of a coloured dot on the solid
phase which is visually red.
Advantages of Dot-ELISA
•It is a simple and rapid assay and can be performed
in the field or poorly equipped laboratories.
•It doesn't require much instrumentation and
results can be observed visually by naked eye.

Dot-ELISA and diagnosis of parasitic diseases
11
•Require minimum basic training to the locally
available technical staff.
paper. Antibodies were detected in experimentally
infected sheep as early as 4 weeks post infection (WPI).
•It is easy to perform and hence Dot-ELISA is a The sensitivity of the most serological assays is
frequently evaluated assay for use in the field satisfactory with crude antigens but specificity remains
(Parija, 1998).
hampered due to cross reactivity with other parasites
infecting ruminants. Efforts are therefore being made to
•Multiple infections can be diagnosed by the use of evolve a putative antigen specific for serodiagnosis of
a single Dot-ELISA strip known as multiple Dot- fasciolosis. Enzymatically active Cathepsin L-cysteine
ELISA.
proteinase from F. gigantica was isolated from adult
Limitations of Dot-ELISA
fluke E/S material and a Dot-ELISA assay utilizing
dipsticks (dipstick-ELISA) was standerdidized for its
•High cost of the assay restricts it wider use.
application in field. The test evidenced positive
reaction at 4 WPI with weekly pooled sera from
•Non-availability of reagents as well as tests experimentally infected sheep (Dixit et al., 2002).
locally so need to import these from the over- These results highlighted for the first time the
seas.
possibilities and advantages of using pure antigen for
the diagnosis of experimental F. gigantica infection.
•Short shelf-lives of the perishable thermo-labile
Weekly pooled sera of infected buffaloes when
enzyme-based reagents.
subjected to dipstick-ELISA revealed antibodies at 2
•It is affected by high environmental temperature
WPI (Dixit et al., 2004). Further, no cross reactivity
and requires proper refrigeration facilities in
was observed with weekly pooled sera of buffaloes and
laboratories.
goats experimentally infected with Paramphistomum
epiclitum (Dixit et al., 2003).
Helminthic diseases
Cathepsin L-cysteine proteinase from F. gigantica was
Fasciolosis: In tropics, fasciolosis mainly caused by also evaluated for its potential in the early prepatent
Fasciola gigantica has been a continuous constraint on detection in bovine calves. F. gigantica infection could
growth, productivity and reproduction of ruminants. be detected 4 WPI using an ELISA, dipstick ELISA and
The diagnosis of fasciolosis has been mainly relied Western blotting with 100% sensitivity. Preliminary
upon the detection of eggs in the feces of infected studies showed that F. gigantica cathepsin L-cysteine
animals. However detection of relatively small number proteinase does not cross-react with P. epiclitum,
of eggs, in comparatively large amount of feces, has Gigantocotyle explanatum and hydatid cyst antigens
been encountered as major bottle-neck in the diagnosis (Sriveny et al., 2006).
with these methods and actually positive animals are
reported false negative. Furthermore, the coproscopic
To test the diagnostic potentiality of the somatic
examination of eggs is impractical solution, when
antigens, ELISA and dot ELISA was standardized using
flukes are yet to mature as adult to discharge eggs,
the sera from experimentally non-infected (group A)
during prepatent period of disease.
and infected (group B) animals. Further, the sensitivity
and the specificity of the assays were evaluated
An alternative answer to overcome above deficiencies employing the field sera from animals of different
associated with coproscopic diagnosis rests in detection parasitic load viz., F. gigantica positive (group C), F.
of anti-Fasciola antibodies and /or circulating antigens gigantica and Gastrothylax crumenifer positive (group
by immunological methods. Immunodiagnosis is D), F. gigantica and G. explanatum positive (group E),
crucial in the control of fasciolosis, as it can detect an a group of sera without F. gigantica but other trematode
early prepatent infection leading to the timely infection (group F), only G. crumenifer positive (group
chemotherapeutic intervention and also preventing the G), only G. explanatum positive (group H), G.
contamination of water bodies with the parasite eggs crumenifer and G. explanatum positive (group I), and
(Dixit et al., 2008). In an effort to develop a more rapid PM negative (group J) collected from slaughterhouses
and economical diagnostic test for fasciolosis, of Bareilly (Uttar Pradesh, India) and Patna (Bihar,
Zimmerman et al. (1985) prepared a crude antigen of India). In plate ELISA, the sensitivity of the antigen and
excretory/ secretory (E/S) preparation of Fasciola the assay was 75.75%, whereas the specificity was
hepatica and adsorbed the mixture on the nitrocellulose 97%, 95%, and 98%, respectively, against G.

12 Gupta et al.
crumenifer, G. explanatum, and mixed infection of G. natural sheep sera having H. contortus infection, 60%
crumenifer and G. explanatum, respectively. In the case sera samples showed solid dot formation whereas in F-1
of Dot-ELISA the sensitivity was 86.5% and specificity fraction 75% of the sera samples showed solid dot
was 92.3%, 94.7%, and 90%, respectively, against G. indicating purified fraction was a more potent antigen.
crumenifer, G. explanatum, and mixed infection of G. Crude E/S and F-1 were also fractionated through SDScrumenifer
and G. explanatum, respectively (Kumar et PAGE. ES antigen revealed polypeptides in the range of
al., 2008).
10–200 kDa of which 26, 32, 60 and 120 kDa were
found more prominent. F-1 fraction on SDS-PAGE
Toxocarosis: Matsumura et al. (1987) conducted a
analysis revealed only four polypeptides of 26, 32, 60,
Dot-ELISA for detecting antibodies against Toxocara
and 120 of which 60 and 120 kDa were found to be most
canis infection in dogs. All assay steps were performed
prominent. Results indicate that the purified fraction of
at room temperature. The well-defined blue spots
ES antigen may be utilized for early diagnosis of
formed on NC paper were evaluated by both visual
haemonchosis. (Prasad et al., 2008)
observation and densitometric reading. The assay was
carried out within 1.5 h.
Trichinellosis: It is a parasitic disease of public health
importance caused by the nematode Trichinella
Dirofilariosis: Matsumura et al. (1988) conducted a
spiralis. Most infections in domestic and wild animals
Dot-ELISA for the detection of antibodies against
go unnoticed. Microscopical examination of muscle
Dirofilaria immitis in dogs. Dirofilarial antigens
biopsy sample may confirm but not necessarily rule out
prepared were directly bound on NC paper set into a
trichinellosis.
microfiltration apparatus to ensure their uniformity. Of
the 23 infected dogs tested, 21 were determined A Dot-ELISA using antigens purified by monoclonal
positive by visual observation of brown-colored spots antibody affinity chromatography was developed for
on NC paper. The positive and negative readings were detecting T. spiralis infection in swine. (Su and
further confirmed by using a densitometer. Sera from Prestwood, 1991).The assay was as sensitive as an
15 non-infected and 17 other parasite-infected dogs ELISA (using E/S products as antigen) and western blot
were all negative, i.e. no false positive readings and analysis and nearly as specific as western blot. The Dotcross-reactivities
were found using this technique. ELISA detected all of 20 low infections (0.08-4.74
Also, a large number of samples could be assayed larvae/gram of diaphragm), most of them by 5–6 WPI.
simultaneously within 1.5 h. Similar results were obtained by Aguilar et al. (2000).
Sera from A. suum infected swine were negative to E/S
Haemonchosis: Detection of infection during
antigen of T. spiralis when analyzed by Dot-ELISA and
prepatency, is of greater importance from the clinical
western blot. The improved specificity was achieved by
point of view, therefore a simplified, field oriented Dotemploying
species-specific denatured antigens. More
ELISA has been developed for the detection of
importantly, the Dot-ELISA was much simpler to
Haemonchus contortus soluble antigen in goat /sheep
perform than western blot analysis.
sera (Sood et al., 1996). Dot-ELISA performed with
immunoaffinity purified somatic antigen could detect Protozoan diseases
infection as early as 1 WPI during pre-patency. Further,
Leishmaniosis: It is a chronic, severe protozoal disease
SDS-PAGE analysis of immunoaffinity purified
of man, dogs and certain rodents characterized by
antigen revealed four polypeptides (26, 32, 60 and 120
cutaneous or muco-cutaneous leisions. The most
kDa) which may be utilized for the detection of H.
reliable diagnostic test for the canine leishmaniosis is
contortus infection in sheep or for immunoprophylaxis
direct observations of the amastigotes in macrophages
(Prasad et al., 2007).
in bone marrow or lymph nodes smears. The
E/S protein of H. contortus was also purified through disadvantage of these methods is that it is sometimes
immunoaffinity chromatography and Dot-ELISA was impossible to detect the parasite in infected animals
performed with crude ES antigen as well as especially in lymph node smears.
immunoaffinity purified fraction (F-1) with
A comparative study was done for the diagnosis of
experimental and natural sera of sheep infected with H.
leishmaniosis by Dot-ELISA and blinded bone marrow
contortus. Solid dot formation took place with four day,
aspirate (Reynaldo et al. 1995). Using visualization of
1, 2 and 3 wpi sera. Dot formation did not take place
amastigotes on bone marrow examination as a priori
with negative control serum and uninfected control
evidence of infection, the Dot-ELISA was found to be
animal serum. When crude E/S antigens was reacted to

Dot-ELISA and diagnosis of parasitic diseases
13
highly sensitive(97%) and specific (100%). In contrast, In addition, the Dot-ELISA may be configured to detect
the physical evaluation had remarkably low sensitivity antibodies or parasite antigen in either micro-titer plates
and specificity. The Dot-ELISAis an excellent assay for for large-batch testing or with dipsticks for small
detection of the canine reservoir of Leishmania, Thus it numbers of determinations. The facilities for
may help to control this debilitating illness by indigenous production and distribution of enzyme
facilitating selective canine elimination.
labeled reagents by a central laboratory and easy
availability of nitrocellulose membrane for coating the
A Dot-ELISA was developed for diagnosing visceral
proteins may reduce the cost of the assay to some extent
leishmaniosis in dogs infected with Leishmania
and make it a popular assay.
infantum (Vercamme et al., 1998). The test procedure
lasted only 30 min. Distinction between Leishmania REFERENCES
positive and Leishmania negative sera was complete at Aguilar FBR, Bautista-Garfias CR, Rojas J, de Nova ME,
a dilution of 1/320, hence there was no cross-reactivity, Rodríguez O I and Martinez-Gómez F. 2000. Experimental
not even with sera from dogs with trypanosomal or swine Trichinellosis: Use of Dot-ELISA and western blot
babesial infections. Therefore, it is concluded that this with excretion/secretion antigens (ES) from infective larvae
to detect anti-Trichinella spiralis antibodies. Rev Latinoam
test has a very high sensitivity and specificity for the
Microbiol 42:57-62
detection of anti-Leishmania antibodies in the dog. The
Dot-ELISA was significantly positive correlated with Camus E and Montenegro James S. 1994. Bovine anaplasmosis
and babesiosis in the Lesser antilles: risk assessment of an
the direct agglutination test (DAT), the indirect
unstable epidemiologic situation. Vet Res 25:313-317.
immunofluorescence test (IFAT), and the slide-ELISA.
Similarly, Dot-ELISA using protein A-peroxidase was Dixit AK, Yadav SC, Saini M and Sharma RL. 2003. Purification
evaluated as a diagnostic test for canine leishmaniasis
and characterization of 28 kDa cysteine proteinase for
immunodiagnosis of tropical fasciolosis. J Vet Parasitol
(Fisa et al., 1997). The test results were in agreement
17:5-9.
with parasitologic diagnosis and indirect
immunofluorescence assay results.
Dixit AK, Yadav SC and Sharma RL. 2002. 28 kDa Fasciola
gigantica cysteine proteinase in the diagnosis of prepatent
Babesiosis: It is a tick transmitted protozoan disease of ovine fasciolosis. Vet Parasitol 109:233-247.
wild and domestic animals. Although microscopic Dixit AK, Yadav SC and Sharma RL. 2004. Kinetics of antibody
detection of Babesia parasites is the most efficient and response using 28 kDa Fasciola gigantica cysteine
cost effective method for the diagnosis of acute proteinase as antigen. Trop Anim Health Prod 36:49-54.
babesiosis, the detection of specific antibody is Dixit AK, Dixit Pooja and Sharma RL. 2008.
recommended for epidemiological surveillance and the Immunodiagnostic/ protective role of Cathepsin L cysteine
assessment of enzootic stability.
proteinase secreted by Fasciola species. Vet Parasitol 154:
177-184.
A reagent conservative Dot-ELISA was developed
Fisa R, Gallego M, Riera C, Aisa MJ, Valls D, Serra T, de
primarily for canine babesiosis caused by Babesia
Colmenares M, Castillejo S and Portus M. 1997. Serologic
canis. Strong agreement was observed between Dot- diagnosis of canine leishmaniasis by Dot-ELISA. J Vet Diagn
ELISA and the conventionally used IFAT Invest 9:50-55.
(Wanduragala et al., 1987). Dot-ELISA has also been
Kumar N, Ghosh S and Gupta SC. 2008.Early detection of
configured for the simultaneous detection of antibodies
Fasciola gigantica infection in buffaloes by enzyme linked
to Babesia bovis, Babesia bigemina and Anaplasma immunosorbent assay and dot enzyme linked immunosorbent
marginale antibodies (Montenegro James et al., 1992). assay. Parasitol Res (in press).
The multidot ELISA is especially suited for sero-
Matsumura K, Kazuta Y, Endo R, Tanaka K, Inoue T and
epidemiological studies in regions where all three Matsuda H. 1988. A rapid microassay for dirofilarial
haemo-parasites are endemic (Camus and Montenegro antibodies by using antigen-coated nitrocellulose paper. Vet
James, 1994). Mattioli et al. (1997) used the multiDot- Parasitol 27 :337-44.
ELISA to survey the seasonal prevalence of tick Matsumura K, Kazuta Y, Endo R, Tanaka K, Inoue T and
transmitted haemoparasites in N'Dama cattle in West Matsuda H. 1987. A rapid detection of toxocaral antibodies
Africa.
by dot enzyme-linked immunosorbent assay (Dot-ELISA) in
CONCLUSIONS
dogs. Zentralbl Baktriol Mikrobiol Hyg 266:580-585.
Mattioli RC, Janneh L, Corr N, Faye JA, Pandey US and Verhulet
The Dot-ELISA is rapid, easy to perform and interpret A. 1997 .Seasonal prevalence of ticks and tick transmitted
reagent conservative, cost effective and field portable. haemoparasites in traditionally managed N'Dama cattle with

14 Gupta et al.
reference to strategic tick control in the Gambia. Med Vet Dot-ELISA for detection of Haemonchus contortus.
Entomol 11:342-348. Helminthologia 33:73-75.
Montenegro James S, Guillen A T and Toro M. 1992. Dot-ELISA
para el diagnostico serologico de la anaplasmosis y
babesiosis bovina. Rev. Ciesstifica FCV de Luz II ,23-29.
Parija SC. 1998. A review of some simple immunoassays in the
serodiagnosis of cystic hydatid disease. Acta Trop 70:17-24.
Sriveny D, Raina OK, Yadav SC, Chandra D, Jayraw AK, Singh
M, Velusamy R and Singh BP. 2006. Cathepsin L-cysteine
proteinase in the diagnosis of bovine Fasciola gigantica
infection. Vet Parasitol 135:25-31.
Su XZ and Prestwood AK. 1991. A Dot-ELISA mimicry western
blot test for the detection of swine trichinellosis. J Parasitol
Prasad A, Nasir, A and Singh N. (2007). Dot-ELISA for the
77:76-82.
detection of preclinical Haemonchus contortus infection in
sheep by using an adult somatic antigen and an Vercammen F, Berkvens D, Brandt J and Vansteenkiste W. 1998.
immunoaffinity purified fraction. J Para Dis 31:22-28.
A sensitive and specific 30-min Dot-ELISA for the detection
of anti-leishmania antibodies in the dog. Vet Parasitol 79:221-
Prasad A, Nasir A and Singh N.2008. Detection of anti-
228.
Haemonchus contortus antibodies in sheep by Dot-ELISA
with immunoaffinity purified fraction of ES antigen during Wanduragala L, Kakoma I, Clabaugh GW, Abeygunawardena I,
prepatency. Ind J Exp Biol 46:94-99.
Levy MG, Ristic M. 1987. Development of Dot-Enzyme
immunoassay for diagnosis of canine babesiosis. Am J Trop
Reynaldo Dietze, Aloisio Falqueto, Luiz Carlos Pedrosa Valli,
Med Hyg 36:20-21.
Temizio Pereira Rodriques, Marcos Boulos and Ralph Corey.
1995. Diagnosis of Canine Visceral Leishmaniasis with a Zimmerman GL, Nelson MJ and Clark CRB. 1985. Diagnosis of
Dot-Enzyme-Linked Immunosorbent Assay. Am J Trop Med ovine fasciolosis by a dot ELISA: A rapid diagnostic
Hyg 53:40-42. technique Am J Vet Res 46:1513-1515.
Sood ML, Kaur G, Parmar A and Kapur J. 1996. Development of

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 15-21
Original paper
J P D
Lymphatic filariasis in Benue State, Nigeria: community
diagnosis using the rapid-format antigen immunochromatographic
card test
1 2
E. A. Omudu and F. C. Okafor
1
Department of Biological Sciences, Benue State University, Makurdi, Nigeria.
2
Department of Zoology, University of Nigeria, Nsukka, Nigeria.
Received 16 July 2007; accepted 10 January 2008
ABSTRACT. The whole blood immunochromatographic card test (ICT) for bancroftian filariasis
was used in a survey conducted in Benue State, Nigeria. Eight hundred and forty five persons in
rural communities were examined, and 264 (31.2 %) of them tested positive for circulating filarial
antigen (CFA). The persons in the age bracket of 40–49 years recorded the highest infection rate; it
2
was significantly higher in males (X = 7.02, df = 1, p < 0.05). Characteristics of the ICT card test
procedure including high sensitivity, promptness and simplicity in field application justify its use
for screening of filariasis. However, the test kits are expensive, and thus surveys based on this
procedure are relatively limited. Further studies designed to use this technique on a wider scale in
different bio-geographical areas in Nigeria are necessary to generate data regarding the
distribution and prevalence of the disease.
Keywords: community diagnosis ICT card test, lymphatic filariasis, Nigeria.
INTRODUCTION
22.1% of its population is thought to be infected
(Michael et al., 1996). Although, mortality from
lymphatic filariasis is low, the disease is the fourth
leading cause of permanent disability and among the
tropical diseases, lies second only to malaria in terms
of disability-adjusted life years (WHO, 2002).
Unprecedented and unplanned urbanization, over-
crowding and deteriorating sanitary conditions in both
urban and rural areas is believed to be exacerbating the
transmission of the disease in Nigeria (Eigege et al.,
2002; Braide et al., 2003; Terranella et al., 2006;
Nwoke et al., 2006).
Lymphatic filariasis, caused by Wuchereria bancrofti
infection, is estimated to infect at least 128 million
persons worldwide. It is a major cause of acute as well
as chronic morbidity and incapacitation among the
poorest 20% of the world's poor in tropical and subtropical
zones of the world (Michael and Bundy,
1997). Lymphatic filariasis is the second leading cause
of permanent and long-term disability in the world,
inflicting serious public health and socio-economic
problem on the people (Terranella et al., 2006).
Nigeria is the third most lymphatic filariasis endemic
country in the world after India and Indonesia, and
Corresponding author: Dr. Edward Agbo Omudu, Department of
Biological Sciences, Benue State University, P M B 102119 Makurdi,
Nigeria. E-mail: eddieomudu@yahoo.com
The global efforts to eliminate lymphatic filariasis as a
public health problem focus on mass drug
administration (MDA), which is currently
recommended in communities known or suspected to
have a prevalence of lymphatic filariasis greater than

16
Omudu and Okafor
1% ( Eigege et al., 2002). To determine community blood for the circulating Og4C3 antigen of W.
eligibility for MDA, community mapping must be bancrofti has raised hope for quick community
conducted either through parasitological or diagnosis (Weil et al., 1997). The ICT test is very
serological investigation. The parasitological sensitive and specific to lymphatic filariasis that
investigation is extremely difficult because it requires detects the infection within minutes, at any time of the
collection of night blood samples; this is particularly day, without the need for laboratory facilities. Several
problematic in many endemic communities in Africa comparative studies have been conducted to compare
with strong religious and superstitious beliefs ICT test with other existing serological diagnostic
(Gyapong et al., 1996; Haddix and Kestler 2000; options (Wamae et al., 1995; Weil et al., 1997;
Braide et al., 2003). Antigen testing, therefore, Simonsen and Dunyo, 1999; Bhumiratana et al., 2000;
became the recognized method of choice for the Eigege et al., 2002). The ICT has been found to be the
detection of W. bancrofti infection. Unlike tests that most appropriate test for the field studies in Africa, and
detect microfilariae, antigen tests can be performed has been adopted as the method of choice for
with blood collected during the day or night. However, community mapping to facilitate implementation of
existing enzyme-linked immunosorbent assay new strategies by the World Health Organization for
(ELISA) test for filarial antigenemia is difficult to the control and elimination of lymphatic filariasis. We
perform in the field, and has resulted in its limited use undertook this study to determine the prevalence of
in endemic countries.
the disease in Benue State, where there is a dearth of
hospital and epidemiological records on the extent and
The development of a rapid immunochromatographic
distribution of the disease.
card test (ICT) that qualitatively tests finger prick
8 o 00 /
8 o 30/
9 00 /
NIGERIA
N A S A R A W A S T A T E
o
8 00
/
A G A T U
G U M A
Benue
State
TARABA
K O G I
o /
7 30
S T A T E
G W E R
MAKURDI
A P A
W E S T
G W E R
O T U K P O E A S T
TARKA L O G O
BURUKU
G B O K O
U K U M
STATE
o /
7 30
O H I M I N I
K A T S I N A - A L A
o /
7 00
U S H O N G O
VANDE-
IKYA K W A N D E
OGBA-
DIBO
ENUGU
OKPOKWU
A D O
O B I
O J U
KONSHISHA
o 9 30 /
N
o /
7 00
STATE
STATE BOUNDARY
L.G. BOUNDARY
WATER WAYS
EBONYI
STATE
8 o 00 /
o /
8 30
NATIONAL BOUNDARY
SELECTED LOCAL
GOVERNMENT AREAS
LGAs NOT COVERED IN THE
R.A.PROCEDURES
CROSS RIVER STATE
o /
9 00
Fig. 1. Local Government Areas where the ICT screening was conducted.
CAMEROON
REPUBLIC
Kilometre
0 15 30 40 45
o
6 o 30 /

Diagnosis of lymphatic filariasis
17
MATERIALS AND METHODS
Description of the study area: Benue State is one of
the 36 states in the Federal republic of Nigeria, a
tropical country on the west coast of Africa. The state
derives its name from River Benue, the second largest
river in the country, and is located in the central region
of the country where it lies between latitude 7° 13 and
7° 49 and longitude 8° 15 and 8° 42 (Fig. 1). The State
covers an area of about 34,059 square km with a
population of over 4.2 million (NPC 2006). Majority
of the inhabitants live in rural agricultural areas and
engage in peasant agriculture, the State's reputation as
the food-basket of the nation is being seriously
jeopardized by the socio-economic consequences of
parasitic diseases.
The study was conducted in 6 out of 23 Local
Government Areas (LGAs) of the State. Permission to
conduct this study was granted by the state Ministry of
Health and Human Services in Makurdi, the
administrative headquarter of the State. Informed
consent was obtained from all the participants after the
explanation of the procedures and the likely benefits
of the study. After explaining the purpose of the study
to the village-chief and the traditional-leadership
council and obtaining their permission and consent, all
the participating adults (16 years of age and older)
were asked to gather at the village Primary Health
Care (PHC) Centre and randomly selected. The
individual verbal consent of the patients was obtained,
following which they were assigned identification
numbers and their names, age, occupation and marital
status were recorded. Then after the collection of
demographic data, ICT was performed as per the
manufacturer's instructions (NOW, ICT filariasis kits,
Binax, Portland, ME, USA). The patient's left index
finger was cleaned with methylated-spirit, and then
punctured by using a sterile lancet. The initial sample
of blood was removed using a cotton swab, and
sufficient fresh blood was then collected to fill a 100-
50
45
percentage positive
40
35
30
25
20
15
10
5
0
Series1
10-19
20-29
30-39
40-49
50-59
60-69
70-above
age-specific results of the ICT card test
in study area
Fig. 1. Age-specific results of ICT in study area.

18
Omudu and Okafor
ql capillary tube. The blood was then transferred from ecological conditions and anthropogenic factors that
the capillary tube to the pad on the ICT card and then may act as risk factors in the areas studied. Drainage
sealed. The results of each ICT were read after 15 min. system and farming practice was similar and 66.2% of
Result was considered positive, when two pink lines the participants were farmers.
appeared on the card's window and negative when
only a single line was seen. Test results with the
DISCUSSION
individual's identification code were recorded on the The assessment of filariasis burden in a community is
patients' diagnostic data sheet.
a pre-requisite for planning, implementing and
Data analysis: Data were entered into a computer and
evaluation of intervention strategies. Sensitivity,
statistical analysis was carried out in Epi Info, version
specificity, speed and simplicity are the important
2002 (Centre for Disease Control and Prevention,
factors considered in assuring efficiency of screening
Atlanta). Chi-Square tests were used to compare and
for bancroftian filariasis. The assessment of field
test for significance.
performance and field applicability of new diagnostic
methods in Africa is an important step towards
RESULTS
stopping transmission. Our findings are similar to
those of others reported in Africa ( Ramzy et al., 1999;
Of the 845 participants, 264 (31.2%) tested positive
Eigege et al., 2002; Terranella et al., 2006) and
for circulating filarial antigen (CFA). The sex of the
elsewhere in Brazil, Sri Lanka and India (Pani et al.,
participant appeared to have a bearing on their
2000; Braga et al., 2003; Weerasooriya et al., 2003).
infection as the males had significantly higher
The overall prevalence of filarial antigenaemia was
infection rate (36.3 %) than the female participants
very high (31.2%), and this is much higher than earlier
2
(25.4%; X = 7.43, df = 1, p < 0.05). Ado LGA recorded reported and far exceeds the World Health
the highest prevalence with 53.7% of sampled Organization's threshold of 1% positivity, for MDA in
population testing positive, whereas Ogbadibo a given community ( WHO, 1998). The high
recorded only 2.6% prevalence (Table I); this endemicity of the disease in Benue State may be as a
2
difference was statistically significant ( X = 9.81, df = result of the deteriorating sanitary conditions, which
6, p < 0.05). The highest age-related prevalence was create breeding sites for mosquitoes, the slow and
recorded in the age group of 40–49 years (Table II); ineffective pace of the distribution and availability of
however, there was no statistically significant Insecticide Treated Bednets (ITBNs) in the Roll Back
difference in the prevalence of antigenaemia Malaria programme and difficulties in diagnosis and
according to age group. Of the 21 villages where the treatment of the disease in many rural health centers.
study was conducted, ICT-detected prevalence ranged
from 0–57.1%. Three villages (Rijo, Ofuke and The bearing of gender in ICT positivity and by
Odiapa in Ado, Apa and Obi LGAs, respectively) had a implication the prevalence of lymphatic filariasis
filarial antigenaemia prevalence rate of > 30%, and observed in this study, is in consonance with other
93.3% of participants were clinically asymptomatic findings in North Central Nigeria and Brazil (Eigege
(showing no visible signs of lymphatic filariasis). et al., 2002; Braga et al., 2006). The reasons for this
Nevertheless, we encountered 6 participants who had gender bearing should generate a lot of research
elephantiasis but ICT gave negative results.
interest because both males and females are equally
exposed to mosquito bites and other risk factors in a
Investigation of treatment history revealed that 68.2% given community. The higher ICT positive results
of the total sample population had taken ivermectin for obtained within the age bracket of 40–49 also agrees
the treatment of onchocerciasis, and 51.1% of those with age-related findings from Nigeria and elsewhere
who had taken ivermectin had been on the treatment (Udonsi, 1986; Meyrowitsch et al., 1995; Surendran
for at least 2 years. Though ICT was easy to use in the et al., 1996; Eigege et al., 2002). The age-related
field and was well-accepted by participating prevalence of lymphatic filariasis in endemic areas
communities; however, we observed 13 cases whose appears to be low among subjects less than 40 years in
card exhibited only one pink line after 15 min in the age and higher in individuals above 40. This is due to
field but a slight pink second line appeared during increased contact between older subjects and the
post-field analysis. These cases were recorded as mosquito vectors, as a result of long exposure during
negative in accordance with the manufacturer's outdoor activities such as farming and fishing.
instructions. There is no marked difference in

Diagnosis of lymphatic filariasis
19
Table I. Prevalence of lymphatic filariasis based on ICT according to sex
Male Female Total
LCA Number Number Percentage Number Number Percentage Number Number Percentage
examined positive (%) examined positive (%) examined positive (%)
Ado 81 44 61.7 66 32 48.5 147 76 55.8
Apa 80 30 37.5 51 9 17.6 131 39 29.8
Gwer 72 29 40.3 29 11 37.9 101 40 39.6
West
Obi 41 22 53.6 69 26 37.7 110 48 43.6
Ogbadibo 52 2 3.84 69 1 1.45 121 3 2.47
Okpokwu 52 10 19.2 43 7 16.3 95 17 17.9
Ushongo 75 19 25.3 76 22 28.9 151 41 27.1
Total 453 156 35.7 403 108 26.8 856 264 31.2

20
Omudu and Okafor
The performance of ICT has been found to be need for complicated laboratory procedures and no
comparable to that of the conventional finger-prick, need for specialized technicians. Its usage in other
night-blood thick smear examination ( Pani et al., parts of Nigeria will help generate the much needed
2000; Braga et al., 2003). Among its several epidemiological information on the distribution and
advantages over the conventional technique are that it prevalence of lymphatic filariasis in the country.
can be performed on the whole-blood sample
collected by finger-prick at any time of the day and the
Conflict of interest: The authors declare that they
results are instant. The ICT can also be stored after use
have no conflict of interest
and then later used to evaluate treatment progression. Authors' contribution: Dr. Omudu Edward and
The ICT has also performed considerably better than Prof. Okafor Fabian both designed the study. The later
many other immunological techniques including conducted the field study, analysis and preparation of
immunoradiometric assay and enzyme linked the manuscript under the supervisionof the former.
immunosorbent assay (ELISA; Weil et al., 1997;
Simonsen and Dunyo, 1999; Ramzy et al., 1999; Acknowledgement: We would like to thank Alves
Weerasooriya et al., 2003). Because the detection of Nina of Binax Incorporate Scarborough, ME, USA,
CFA is a useful non-microscopic method for the manufacturers of ICT filariasis test-kits for
diagnosis of bancroftian filariasis that is currently facilitating the purchase of the test kits and helping
making the transition from the research laboratory to with the logistics of sending them to Nigeria.
the field, the ICT surpasses these other immunological
techniques in field application and simplicity.
REFERENCES
Gyapong et al. (1998) and Eigege et al. (2002) have all Bhumiratana A, Koyadun S, Rojanapremsuk J and
Suvannadabba S. 2000. Diagnosis of Wuchereria bancrofti
reported positive correlation between ICT results and
infection in endemic populations: diagnostic approaches to
community hydrocoele rates in Africa.
control and elimination. Mandihol J 7:101-107.
Notwithstanding the brilliant performance of ICT, its Braga C, Dourado MI, Ximenes RA, Alves L, Brayner F, Rocha
large-scale field use in control programmes in A and Alexander N. 2003. Field evaluation of the whole
endemic countries (especially in poor African and blood immunochromatographic test for rapid bancroftian
Southeast Asian countries) will require a
filariasis diagnosis in the Northeast Brazil. Rev Inst Med
Trop S Paulo 45: 125-129.
downward review of its cost. As at the end of
2006, only very few countries in Africa have been able Braide EI, Ikpeme B, Edet E, Atting I and Kale OO. 2003.
to conduct their lymphatic filariasis mapping exercise, Preliminary observations on the occurrence of lymphatic
filariasis in Cross Rivers state, Nigeria. Nigeria J Parasitol
a crucial component of national eradication
24:9-16.
programmes. The slow pace of mapping filariasis is as
a result of the high cost of ICT; ICT survey in just one Eigege A, Richards FO, Blaney DD, Miri ES, Gontor I, Ogah G,
village in Nigeria is estimated to cost well over US $ Umaru J, Jinadu MY, Matthias W, Amadiegwu S and
Hopkins DR. 2002. Rapid Assessment for lymphatic
100,000 (Eigege et al., 2002). In India, the cost of an
filariasis in central Nigeria: a comparison of the
ICT kit alone is between US $ 1.05 and US $ 1.29, it is
immunochromatographic card test and hydrocoele rates in
estimated that 500 tests will cost US $ 120 (Pani et al., an area of high endemicity. Am J Trop Med Hyg 68:643-646.
2000). In Sri Lanka, cost analysis of the kit showed
Gyapong M, Gyapong JO, Adjei S, Vlassoff C and Weiss M.
that ICT costs US $ 2.75 per unit compared to thick-
1996. Filariasis in Northern Ghana: some cultural beliefs
blood film, which is US $ 0.30 (Braga et al., 2003). and practices and their implications for disease control. Soc
The World Health Organization may consider Sci Med 43:235-242.
subsidizing the cost of ICT kits and facilitating
Gyapong JO, Webber RH, Morris J and Bennetts S. 1998.
improved access to the technology for poor countries,
Prevalence of hydrocoele as a rapid diagnostic index for
where the disease is most endemic. The other area of lymphatic filariasis. Trans R Soc Trop Med Hyg 98:40-43.
challenge is the storage requirement, as the ICT cards
need to be stored at 2°C to 8°C, and this may be Haddix A and Kestler A. 2000. Lymphatic filariasis: economic
aspects of the disease and programme for its elimination.
difficult in many rural areas in the tropics.
Trans R Soc Trop M Hyg 94:592-593.
Despite these challenges, the features of the ICT
proved to be extremely advantageous: its high
sensitivity, the ability to offer prompt diagnosis, no
Nwoke BEB, Dozie INS, Jiya J, Saka Y, Ogidi JA, Istifanus WA,
Mafiana CF, Oyene U, Amali O, Ogbu-Pearce P and Nuttal I.
2006. The prevalence of hydrocoele in Nigeria and its

Diagnosis of lymphatic filariasis
21
implication on mapping of lymphatic filariasis. Nigerian J
Parasitol 27:29-35.
Michael E, Bundy DAP and Grenfell BT. 1996. Re-assessing
the global prevalence and distribution of lymphatic
filariasis. Bull WHO 75:491-503.
Michael E and Bundy DAP. 1997. Global mapping of
lymphatic filariasis. Parasitol Today 13:471-476.
Ramzy RMR, Helmy H, El-Lethy AST, Kandil AM, Ahmed ES,
Weil GJ and Faris R. 1999. Field evaluation of a rapidformatkit
for the diagnosis of bancroftian filariasis in Egypt.
East Mediterr Health J 5:880-887.
Terranella A, Eigege A, Gontor P, Dagwa P, Damishi S,
Blackburn B, McFarland, D. Zingeser, J. Jinadu, M.Y., Miri,
E. and Richards F.O. (2006). Urban lymphatic filariasis in
central Nigeria. Ann Trop Med Parasitol 100:1-10.
Udonsi JK. 1986. The status of human filariasis in relation to
clinical signs in endemic areas of the Niger Delta. Ann Trop
Med Parasitol 80:425-432.
Weil GJ, Lammie PJ and Weiss N. 1997. The ICT filariasis test:
a rapid–format antigen test for the diagnosis of Bancroftian
filariasis. Parasitol Today 13:401-404.
Weerasooriya MV, Itoh M, Kimura E and Fujimaki Y. 2003.
Human infection withWuchereria bancrofti in Matara, Sri
Lanka: The use of ELISA to detest 1g G4
in urine and ICT and
test to detect antigen in blood. Ann Trop Med Parasitol
97:179-185.
Pani SP, Hoti SL, Elango A, Yuvaraj J, Lall R and Ramaiah KD.
2000. Evaluation of the ICT whole blood antigen card test to
detect infection due to nocturnally periodic Wuchereria
bancrofti in south India. Trop Med Int Health 5:359-363.
World Health Organization. 1998. Report of a WHO Informal
Consultation on Epidemiologic Approaches to Lymphatic
Filariasis Elimination: Initial Assessment, Monitoring and
Certification. Geneva: WHO.
Meyrowitsch DW, Simonsen PE and Makunde WH. 1995.
Bancroftian filariasis: analysis of infection and disease in
five endemic communities in north-eastern Tanzania. Ann
Trop Med Parasitol 89:653-663.
Surendran K, Pani SP, Soudarssanane MB, Spinivasa DK,
Bordolai AI and Subramania S. 1996. Natural history, trend
of prevalence and spectrum of manifestations of bancroftian
filarial disease in Pondicherry (South India). Acta Tropica
61:9-18.
Simonsen PE and Dunyo SK. 1999. Comparative evaluation of
three new tools for diagnosis of bancroftian filariasis based
on detection of specific circulating antigens. Trans R Soc
Trop Med Hyg 93:278-282.
Wamae CN, Lammie PJ and Muttunga JN. 1995. Bancroftian
filariasis: profile of serum antifilarial antibody and
circulating parasite antigen. East Afr Med J 72:492-494.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 22-29
Original paper
J P D
Purification and characterization of Plasmodium berghei
glutathione reductase
1 2 1
G. Kapoor , U. Bagai and H. S. Banyal
1
Department of Biosciences, Himachal Pradesh University, Shimla.
2
Department of Zoology, Panjab University, Chandigarh.
Received 18 January 2008; accepted 31 March 2008
ABSTRACT. Glutathione (GSH) is a major low molecular mass thiol present in many organisms
and helps to maintain a reduced intracellular environment. GSH and glutathione reductase (GR)
act as an antioxidant defense system by protecting the cell from the damage caused by free-radicals.
+
Glutathione reductase (glutathione: NADP oxidoreductase E.C. 1.8.1.7; GR) was analysed in
rodent malaria parasite, Plasmodium berghei and purified through ammonium sulphate
fractionation and Sephadex G-200. Cell-free P. berghei showed specific activity of 0.617±0.08
units/mg protein. Maximum GR activity was observed in cytosolic fraction of P. berghei. The
molecular weight of parasite enzyme was estimated to be 25 kDa and it was maximally active at pH
6.8 and at 25–40°C. Km and Vmax values for oxidised glutathione (GSSG) substrate were 0.02 mM
and 0.5 mM, respectively. Methylene blue non-competitively inhibited the enzyme and its Ki was 0.4
mM.
Keywords: enzyme characterization, glutathione reductase, malaria parasite, Plasmodium berghei
INTRODUCTION
Plasmodium has been considered very sensitive to
oxidative stress. During the erythrocytic stages of its
life cycle, the parasite is subjected to an increased
oxidative attack by reactive oxygen species (ROS)
produced by the parasite, red cells and host immune
response (Atamna and Ginsburg, 1993). To combat
this stress the parasite relies upon antioxidant
enzymes like glutathione reductase, thioredoxin
reductase, superoxide dismutase, glutathione
transferase etc. The tripeptide glutathione (GSH) is a
major low molecular mass thiol in almost all
eukaryotes with various cellular functions. GSH
Corresponding author: Prof. H. S. Banyal, Department of
Biosciences, Himachal Pradesh University, Shimla-171 005,
India. Email: hsbanyal@yahoo.co.in
regulates the metabolism of proteins and their
activities through thiol-disulphide exchange, plays a
role in antioxidant defense and in maintaining a
reduced environment of the cytosol (Becker et al.,
2003).
Glutathione reductase (GR; E.C. 1.8.1.7), a member of
the pyridine nucleotide disulphide oxidoreductase
family of flavoenzyme catalyses the reduction of
oxidised glutathione (GSSG) to reduced form (GSH)
in the presence of nicotinamide adenine di-nucleotide
phosphate reduced (NADPH). Reduced glutathione is
used in the detoxification of xenobiotics, protecting
thiol groups of intracellular proteins, scavenging
hydrogen peroxide and other organic peroxides, and
helps in maintaining a reduced environment inside the
cytosol. GR has been studied in human erythrocytes
(Worthington and Rosemeyer, 1974; Le Trang et al.,

Plasmodium berghei GR
23
1983), bovine erythrocytes (Erat et al., 2003), sheep determined spectrophotometrically in a double beam
liver (Ulusu et al., 2005) and Plasmodium falciparum spectrophotometer (Perkin Elmer) at room
(Krauth-Siegal et al., 1996). In the present study GR of temperature according to the modified method of
P. berghei has been purified and some of its Worthington and Rosemeyer (1974). The 2 ml assay
characteristics have been investigated.
system contained 0.01M potassium phosphate buffer,
MATERIALS AND METHODS
pH 7.2, 0.2 mM EDTA, 0.1 mM NADPH and an
appropriate volume of enzyme extract, and the
Parasite: P. berghei (NK 65) was maintained in reaction was initiated by the addition of 0.1 mM GSSG
BALB/c mice, and the course of parasitaemia was at room temperature. The oxidation of NADPH
monitored in Giemsa-stained thin blood smears. resulting in decrease in absorbance was monitored at
Normal or P. berghei-infected (about 50% 340 nm. One enzyme unit is defined as the oxidation of
parasitaemia) mice were anaesthetized with diethyl 1µ mol NADPH/min under the assay conditions.
ether and blood was collected in citrate saline.
Protein was determined according to Lowry et al.
(1951), and specific activity of GR was units of
Enzyme extract: The citrated-blood from normal or enzyme/mg protein.
P. berghei-infected mice was centrifuged at 1,000 x g
o
for 10 min at 4 C. The plasma was then aspirated, and Ammonium sulphate fractionation: The cell-free
the pellet suspended in equal volume of phosphatewith
ammonium sulphate in steps of 0%-20%, 20%-
parasite homogenate was subjected to precipitation
buffered saline (PBS, 0.01M), pH 7.2, and loaded onto
a CF-11 cellulose (Whatmann) column (1.5 cm x 20 40%, 40%-80% and 80%-100% saturation. The
cm) to remove leukocytes (Sharma and Banyal, 2007). precipitates of each cut were dissolved in a minimum
The eluted leukocyte-free blood was centrifuged at volume of 50 mM potassium phosphate buffer pH 7.2
1,000 x g for 10 min at 4°C. A part of the pellet was and dialyzed at 4ºC using the same buffer containing
used for total erythrocytes, and the remaining pellet 1mM EDTA with 2–3 changes of buffer.
was suspended in an equal volume of saponin in PBS Gel filtration on Sephadex G-200: Dried Sephadex
o
(0.2% w/v), pH 7.2 and incubated for 30 min at 4 C G-200 (Sigma) was swollen in distilled water for 72 h
with intermittent mixing. The suspension was then at 4°C. A glass column was filled with Sephadex G-
o
centrifuged at 15,000 x g for 20 min at 4 C. The 200 (1.5 cm x 20 cm) and a flow rate of 12 ml/h was
haemolysate thus obtained was aspirated, while maintained. The column was equilibrated with 50 mM
erythrocyte membranes from normal mice were potassium phosphate buffer, pH 7.2 containing 1mM
washed thrice with PBS. In case of infected animals, EDTA until the final absorbance difference became
the erythrocyte membranes overlying cell-free zero at 280 nm. The dialyzed sample was loaded onto
parasites were separated. Both the preparations were the column and elutions were collected in 1 ml volume
washed thrice with PBS (Banyal and Fitch, 1982). The at 4°C. In each fraction enzyme activity and protein
preparation from normal mice yielded total were determined.
erythrocytes, haemolysate and erythrocyte
membranes, and infected blood was fractionated in the Characterization of GR: Sodium dodecyl sulphate
above three components and cell-free parasites also. polyacrylamide gel electrophoresis (SDS-PAGE).
The total erythrocytes, haemolysate, erythrocyte Purified enzyme was electrophoretically
membranes and cell-free parasite were suspended in characterized using sodium dodecyl sulphate
an appropriate volume of 0.01M PBS, pH 7.2, polyacrylamide gel electrophoresis by the method of
homogenized in Potter Elvehjem homogenizer at 4ºC Laemmli (1970) in a mini vertical slab gel apparatus
o
and centrifuged at 1,000 x g for 10 min at 4 C, and the (Genei, Bangalore) as given by Banyal and Inselburg
supernatant was used as an enzyme extract.
(1985) employing 3% stacking and 10% separating
gels. The gels were stained in silver nitrate (Merril et
Subcellular fractionation: Cell-free P. berghei was al., 1981). The pH for optimum activity of P. berghei
homogenized at 4°C in pre-chilled 0.25 M sucrose GR was determined by using .01M potassium
solution. The resulting homogenate was subjected to phosphate buffer for pH 5.5–8.0.
differential centrifugation according to Banyal et al.
(1979) and different fractions were obtained.
To study the effect of temperature on GR activity,
enzyme alone was first incubated for 10min at 0°C,
Enzyme assay: Glutathione reductase activity was 25°C, 40°C, 80°C and 100°C (boiling water bath).

24 Kapoor et al.
After incubation at desired temperature the sample was also noticed in other fractions.
was transferred to ice bath so that further destruction
of enzyme could be prevented. This treated enzyme
P. berghei GR protein was mainly precipitated with
was then assayed.
40%-80% of ammonium sulphate resulting in 8.3 fold
purification of the enzyme (Table III). When the
Km and Vmax values for oxidised glutathione (GSSG) precipitated protein was dialyzed and loaded on to
substrate was calculated from Lineweaver-Burk plot. Sephadex G-200 column the enzyme was eluted in
Enzyme activity was assayed at different GSSG fractions 11 to 17 (Fig. 1) with a peak in fraction 13.
concentrations with a fixed concentration of NADPH. This fraction contained 2.19 units/mg of enzyme.
There was a 14.5 fold purification of the enzyme by
Ki for methylene blue was determined by incubating
Sephadex G-200.
purified enzyme with or without Methylene blue for 5
min at room temperature and then assayed.
SDS-PAGE analysis showed that molecular weight of
RESULTS
P. berghei GR was 25kDa (Fig.2). The enzyme was
significantly active between pH 6.6 to 7.0 with
P. berghei exhibited significant activity of glutathione optimum activity at 6.8 (Fig. 3). Preincubation of
reductase (Table I). Parasite enzyme activity was enzyme at different temperatures showed that the
about three fold higher compared to host erythrocytes. enzyme was active at lower temperatures while no
Parasitized haemolysate contained more activity of activity observed when the protein treated at 100°C
enzyme than the normal haemolysate. The host (Table IV). The Vmax and Km for GSSG were 0.5mM
erythrocyte membranes were without any enzyme and 0.02mM respectively (Fig. 4). Methylene blue
activity.
non-competitively inhibited GR-GSSG enzyme
substrate reaction with Ki of 0.4mM (Fig. 5).
The enzyme was mainly confined to the cytosolic part
of the parasite (Table II) though a very little activity DISCUSSION
Table I. Activity of glutathione reductase in normal and P. berghei-infected erythrocytes and their fractions
Erythrocyte fraction Normal P. berghei-infected
(U/mg)
(U/mg)
Total 01781+ _ 0.02 0.186 + _ 0.02
Haemolysate 0.170 + _ 0.02 0.342 +_ 0.40
Erythrocyte membranes Nil Nil
Cell-free parasite - 0.617 +_ 0.08
Results are mean + _ SD of three experiments.
Table II. GR activity in subcellular fractions of P. berghei
Sediment
Specific activity
(U/mg)
None (homogenate) 0789 + _ 0.11
600xg 0.160 + _ 0.02
10,000xg 0.370 +_ 0.19
24,000xg 0.02 + 0.03
105,000xg
Supernatant 3.7 +_ 0.53
Results are mean +_ SD of three experiments.
Nil

Plasmodium berghei GR
25
Tabel III. Purification of P. berghei GR by ammonium sulphate and Sephadex G-200
Fraction Volume Activity Protein Specific Purification
(ml) (U) (mg/ml) activity (X/fold))
(U/mg)
Crude 14 1.07 1.06 0.3369 1.0
0%-20% 2 Nil 10.0 Nil Nil
20%-40% 2.5 Nil 5.7 Nil Nil
40%-80% 3 8.6 3.0 2.8 8.3
80%-100% 1.5 Nil 0.500 Nil Nil
Sephadex
eluate
11 1 0.064 0.387 0.165 0.4
12 1 0.321 0.354 0.906 2.6
13 1 0.707 0.322 2.19 6.5
14 1 0.385 0.516 0.746 2.2
15 1 0.257 0.580 0.433 1.2
16 1 0.192 0.419 0.458 1.3
17 1 0.064 0.480 0.132 0.3
Table IV. Effect of temperature on activity of P. berghei GR
Temperature
Specific activity (U/mg)
-4°C 0.187
0°C 0.187
25°C 0.200
40°C 0.200
60°C 0.080
80°C 0.026
100°C 0.00
The undesirable biological effects of oxidative agents oxidative stress since the parasite lacks catalase and
like free radicals and reactive oxygen species are GSH peroxidases. Maximum activity of P. berghei GR
eliminated by enzymatic and non-enzymatic was observed in cytosolic part of the parasite. GSH
antioxidant defense systems. P. berghei parasitized dependent decomposition of toxic FP IX occurs in the
host cells contained GR activity while the erythrocyte cytosol, hence maximum GR activity observed in
membranes lacked this enzyme. Increased GR activity cytosolic fraction may be due to such requirement. A
in P. berghei-infected erythrocytes indicates that GSH linked thioredoxin-glutathione system in the cytosolic
is not taken up from the host but there is GSH synthesis and mitochondrial compartment has been
due to the action of glutathione cycle in parasite. characterized in helminths also (Salinas et al., 2004).
Krauth-Siegal et al (1996) also reported higher GR
activity in P. falciparum-infected erythrocytes
SDS-PAGE of purified GR revealed a subunit
compared to normal host erythrocytes. Parasite's own
molecular mass of 25kDa, however, P. falciparum GR
GR activity enables the parasite to maintain its
is reported to be a dimer protein of 105kDa (Krauth
reducing environment efficiently under enhanced
Siegal et al., 1982) with a subunit mass of 54.7kDa. h

26 Kapoor et al.
2.5
2.5
Protein (mg/ml)
2
1.5
1
0.5
2
1.5
1
0.5
Specific activity (U/mg)
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Fraction number
0
Protein (mg/ml)
Specific activity (U/mg)
Fig. 1. Purification of glutathione reductase from cell-free P. berghei using Sephadex G-200.
kDa
97.4
66
43
29
Fig. 2.
1 2 3 4
SDS-PAGE of GR purified from P. berghei.
Lane 1 = Protein standard
Lane 2 = Cell-free parasite homogenate
Lane 3 = Fraction 12
Lane 4 = Fraction 13

Plasmodium berghei GR
27
0.2
Specific activity (U/mg)
0.16
0.12
0.08
0.04
0
6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
pH
Fig. 3. Effect of pH on activity of GR purified from P. berghei.
Fig. 4. Effect of different concentrations of GSSG on activity of P. berghei GR.

28 Kapoor et al.
Fig. 5. Ki determination of P. berghei GR using methylene blue.
Taking into account the prosthetic group FAD P. Intraerythrocytic oxidative stress to parasite is
falciparum GR was found to be a homodimer of combated with the help of antioxidant enzymes, which
111kDa (Krauth Siegal et al., 1996) while human GR are essential for their survival. Disruption of
is a dimer with a subunit mass of 52.3kDa (Becker et antioxidant enzymes like GR is a feasible way to
al., 1991). The variation in enzyme molecular weight interfere with parasite development during
of rodent and human malaria parasite may be due to erythrocytic schizogony and such enzymes may be
different species or monomer and dimer nature of GR. exploited in formulation of antimalarials to disrupt the
P. berghei GR showed maximum activity in the propagation of malaria parasite.
temperature range of 25ºC to 40ºC while at 80ºC GR
activity was very little and the enzyme was maximally
ACKNOWLEDGEMENTS
active at pH 6.8. These results are similar to GR from Mr. Gaurav Kapoor is grateful to ICMR, New Delhi
P. falciparum and human red blood cell GR (Krauth for providing JRF-SRF.
Siegal et al., 1996).
REFERENCES
Km of P. berghei GR for GSSG is 0.02mM and Vmax
Atamna H and Ginsburg H. 1993. Origin of reactive oxygen
0.5mM. Ulusu et al. (2005) reported Km value of
species in erythrocytes infected with Plasmodium
0.02mM for GSSG and 0.025mM for NADPH while falciparum. Mol Biochem Parasitol 61:231-241.
Vmax was 0.255mM and 0.266mM for GSSG and
Banyal HS and Fitch CD. 1982. Ferriprotoporphyrin IX
NADPH respectively in sheep liver GR. Schistosoma
binding substances and the mode of action of chloroquine
digitata GR has been found to have Km value of against malaria. Life Sciences 31:1137-1142.
27.4µM for GSSG (Muller et al., 1995). Methylene
blue is a specific inhibitor of GR. Methylene blue nonof
parasite inhibitory Plasmodium falciparum monoclonal
Banyal HS and Inselburg J. 1985. Isolation and characterization
competitively inhibited P. berghei GR with Ki of
antibodies. Am J Trop Med Hyg 34:1055-1064.
0.4mM.

Plasmodium berghei GR
29
Banyal HS, Pandey VC and Dutta GP. 1979. Subcellular Le Trang N, Bhargava KK, Cerami A. 1983. Purification of
fractionation and localization of marker enzymes of glutathione reductase from gerbil liver in two steps. Anal
erythrocytic stages of Plasmodium knowlesi. Indian J Biochem 133:94-99.
Parasitol 3:9-14.
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. 1951.
Becker K, Kanzok SM, Lozef R, Fischer M, Schirmer RH and Protein measurement with Folin phenol regent. J Biol Chem
Rahlfs S. 2003. Plasmoredoxin, a novel redox-active 193:265-275.
protein unique for malarial parasites. Eur J Biochem
270:1057-1064.
Merril CR, Goldman D, Sedman SA and Ebert HM. 1981.
Ultrasensitive stain for protein in polyacrylamide gel shows
Becker K, Krebs B and Schirmer RH. 1991. Protein-chemical regional variation in cerebrospinal fluid proteins. Science
standardization of the erythrocyte glutathione reductase 211:1437-1438.
activation test. Application to hyperthyroidism. Int J Vit
Nutr Res 61:180-187.
Muller S, Walter RD and Fairlamb AH. 1995. Differential
susceptibility of filarial and human erythrocyte glutathione
Erat M, Sakiroglu H and Ciftci M. 2003. Purification and reductase to inhibition by trivalent organic arsenical
characterization of glutathione reductase from bovine melarson oxide. Mol Biochem Parasitol 71:211-219.
erythrocytes. Prep Biochem Biotechnol 33: 283-300.
Salinas G, Selkirk ME, Chalar C, Maizels RM and Fernandez C.
Krauth-Siegal RL, Blatterspiel R, Saleh M, Schulz GE, 2004. Linked thioredoxin-glutathione systems in
Schirmer RH and Untuchi-Grau R. 1982. Glutathione platyhelminths. Trends Parasitol 20(7):340-346.
reductase from human erythrocytes. The sequence of the
NADPH domain and of the interface domain. Eur J Biochem
Sharma SK and Banyal HS. 2007. Glutathione synthetase in
121,259-267.
Plasmodium berghei. J Parasit Dis 31:33-37.
Krauth-Siegal RL, Muller JG, Lottspeich F and Schirmer RH.
1996. Glutathione reductase and glutamate dehydrogenase
of Plasmodium falciparum, the causative agent of tropical
malaria. Eur J Biochem 235:345-350.
Laemmli UK. 1970. Clevage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-
685.
Ulusu G, Ciftci M, Erat M, Sakiroglu H and Bakan E. 2005.
Purification and characterization of glutathione reductase
from sheep liver. Turk J Vet Anim Sci 29:1109-1117.
Worthington DJ and Rosemeyer MA. 1974. Human glutathione
reductase: purification of crystalline enzyme from
erythrocytes. Eur J Biol 48:167-177.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 30-33
Original paper
J P D
Human sparganosis in Sri Lanka: a case report and
review of the cases
R. L. Ihalamulla, H. Gunatilaka and S. D. Fernando
Department of Parasitology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka.
Received 27 February 2008; revised 22 April 2008; accepted 15 May 2008
ABSTRACT. The occurrence of Spirometra spp. larvae infection is rare in Sri Lanka; only five cases
have been documented so far. Herein, we report a new case of sparganosis in a three year old girl. In all
these cases, there is no history of eating raw flesh of paratenic hosts or application of poultices from
such hosts. Drinking water contaminated with Cyclops, infected with cestode larvae, seems to be the
source of infection in Sri Lanka.
Keywords: sparganosis, Sri Lanka.
INTRODUCTION
Human sparganosis is a zoonotic infection, usually of
subcutaneous tissue, caused by plerocercoids or second
stage larvae (sparganum) of the genus Spirometra
(Pseudophyllidea:Diphyllobothriidae). The adults of
these tapeworms are natural intestinal parasites of some
species of domestic and wild canids and felids. The life
cycle involves two intermediate hosts. The first one is a
freshwater crustacean (copepod) such as Cyclops, and
the second one, which is a paratenic host, belongs to
different kinds of vertebrates (snakes, frogs,
hedgehogs, fowl, pigs and man). Humans are accidental
hosts in whom a subcutaneous nodule, which contains
the larval tapeworm would appear (Chandler, 1955).
The surgical removal of worm cures the condition.
However, the infection can cause serious damage when
vital organs such as brain or eye are involved, especially
if larvae have long-lived and are invasive. Human
sparganosis is rare, and about 300 cases have been
reported the world-over (reviewed by Norman et al.,
1980; Kron et al., 1991). The majority of the cases have
been reported from the American countries (Norman et
Corresponding author: Dr. S. D. Fernando, Department of
Parasitology, Faculty of Medicine, University of Colombo,
Kynsey Road, Colombo 08, Sri Lanka. Email: deepfern@slt.lk
al., 1980; Kron et al., 1991) and the Far East (reviewed
by Ausayakkun et al., 1993; Tsou and Huang, 1993).
Case reports from South Asia are rare (reviewed by
Wijesundera et al., 1997). From Europe, eight cases
have been published (Pampiglione et al., 2003; Rehak
et al., 2006). Herein, we report the sixth case from Sri
Lanka.
Case report: In March 2007, a three year old girl from
the District of Gampaha, in the Western Province of Sri
Lanka, presented at the Base Hospital, Gampaha, with a
one month old lump in the anterior abdominal wall. The
lump was excised under general anaesthesia. In the
middle of the lump, a worm was found and this was
removed and sent to the Department of Parasitology,
Faculty of Medicine, University of Colombo, for
identification.
Description of the worm: On gross examination the
worm was found to be flat, solid and creamy-white in
colour, and was in three pieces. It showed
pseudosegmentation. The anterior or the head-end was
broader (1.2 mm) than the rest of the body (456 µm).
There was a subtle invagination at the anterior margin
of the head. Haematoxylin and eosin (H & E) stained
sections of the body showed a typical cestode cuticle,
subcuticular cells and parenchymatous tissue which

Review of sparganosis in Sri Lanka
31
filled the space with longitudinal muscle bundles surrounded by tracts of suppurative inflammation.
scattered at random (Fig. 1). A few thin walled
excretory ducts were also observed. According to the
The second case reported was a 28 year old woman
description given by Chandler, 1955, the worm was
presented with a swelling of the left upper arm of two
identified as a plerocercoid larva (sparganum) of
months duration. The lump was non-tender and of 2 cm
Spirometra spp.
diameter. It was excised surgically. H & E stained
sections of the lump showed a cestode larva surrounded
DISCUSSION
by the host-tissue. The presence of calcareous
corpuscles, with no concentric layers, suggested that
All the six cases of human sparganosis reported from
this larva was probably a tetrathiridium of a
Sri Lanka involved the subcutaneous tissue, except for
Mesocestoides worm. Mesocestoides worms have been
a single case of cerebral sparganosis (Alibhoy et al.,
reported from cats and dogs in Sri Lanka (Seneviratna,
2007). The first two cases of human sparganosis from
1955; Seneviratna and Mahalingam, 1962).
Sri Lanka caused by plerocercoid larvae were reported
in 1997(Wijesundera et al., 1997). The larvae were seen Subsequently, two more cases involving the anterior
in tissue sections of subcutaneous lumps in the left abdominal wall were reported. The first of these was
groin and left upper arm, respectively. In the first case, a that of a 40 year old male from the District of Kurunegla
45 year old man from the District of Kegalle was seen at in the north-western province, who presented with a
the General Hospital, Kegalle, with a painless lump in painful tender lump of about five years duration
the groin of 3 months duration. On examination, a non- (Dissanaike et al., 2001). During surgery, a preliminary
tender lump of about 4 cm diameter was seen and an diagnosis of an omental protuberance from
excision biopsy was done. H & E-stained section of the paraumbilical hernia was made by the surgeon. An
lump showed a solid cestode larva that measured 1.1 ovoidal subcutaneous granuloma with a length of 10
mm in width. The parasite was in the subcutaneous fat cm, a width of 8.7 cm and a thickness of about 2 cm was
Fig. 1. Section of bodyof larva; bar = 200 µm

32 Ihalamulla et al.
dissected out. From the middle of the granuloma a live
worm crept out. There was no evidence of hernia. The
worm was a plerocercoid larva (sparganum) of
Spirometra spp, as identified by external morphology
and histological studies.
The next case was also reported by Dissanaike et al.,
2001 and involved a young female of 23 years from the
District of Kalutara in the western province. The patient
presented with a migratory lump of 4 months duration
with associated pain, in the anterior abdominal wall to
the left of the umbilicus. She also complained of
nausea, fever and loss of appetite for the same period.
Surgery of the lump revealed an oval-shaped 3 cm
long and 2 cm wide mass of fibrous and fatty tissue.
When this was opened a whitish, thread like worm
emerged. This worm too was a sparganum of
Spirometra spp., identified in the same way as in the
previous case.
The fifth case was that of a 13 year old boy who
presented with three focal motor seizures which
involved the right arm and leg, over a period of three
days (Alibhoy et al., 2007). An enhancing mass lesion
with perilesional oedema in the left frontal lobe was
observed in neuroimaging. The patient's blood reports
were normal. Craniotomy and excision of the lesion
was performed. The child had remained seizure-free on
carbamazepine therapy since surgery. H & E-stained
sections of the lesion showed a granulomatous reaction
consisting of a necrotic area surrounded by histiocytes,
neutrophils, plasma cells and eosinophils. Sections of a
typical cestode larva showed prominent calcareous
bodies with concentric laminations. This was also
probably a sparganum of Spirometra spp.
The adult worms of these tapeworms, which are natural
parasites of cats and dogs, shed eggs which hatch into
coracidia, in water. These eggs, if ingested by
crustaceans such as Cyclops, develop into the
procercoid or the first larval stage. Ingestion of infected
Cyclops by the second intermediate hosts such as frogs,
snakes, fowl and some mammals including humans,
give rise to the second larval stage, the plerocercoid
larva or the sparganum. The tapeworm species of
Spirometra (Pseudophyllidae) and Mesocestoides
(Cyclophyllidea) have been reported in dogs and cats in
Sri Lanka (Seneviratna, 1955; Seneviratna and
Mahalingam, 1962). The larval stages of these worms,
spargana of Spirometra and tetrathiridia of
Mesocestoides cannot be distinguished, macroscopically.
However, in transverse sections, the headend
of the sparganum shows bothrial slits, whereas that
of the tetrathiridium shows suckers. Further, the
sections of the body show different arrangements in
parenchymal longitudinal musculature. In sparganum,
the bundles of musculature are scattered at random in
the parenchyma, and in tetrathiridium these form an
elliptic band (Andersen, 1983). The presence or the
absence of concentric rings in calcareous corpuscles
also helps in differentiating the two larvae; the rings
being absent in the Mesocestoides larvae. In the second
case reported from Sri Lanka (Wijesundera et al.,
1997), the larva had no concentric rings in the
calcareous corpuscles and it was probably a
tetrathiridium. If so, this would be the first report of a
human infection by larva of Mesocestoides spp.
However, it was not possible to confirm the diagnosis as
the scolex of the larvae could not be demonstrated.
Spargana of Spirometra erinacei (=Mansoni) is known
to infect humans in Asia and S. mansonoides in the
Americas. S. erinacei had been reported from dogs in
Sri Lanka (Seneviratna, 1955). The human sparganosis
cases reported from Sri Lanka were probably caused by
spargana of S. erinacei.
Humans can rarely be final hosts of Spirometra spp.
They can become infected in three known ways: (a) by
drinking water contaminated with parasitized
copepods, (b) by consuming raw or undercooked meat
of a parasitized second intermediate host, and (c) by
direct entry of plerocercoid larva through the skin or the
mucosa (eye, vagina) or through an open wound, when
poultices made of flesh of intermediate hosts such as
frogs or snakes are applied as local remedies (Miyazaki,
1991). The application of poultices is common practice
in Vietnam and Thailand (Ausayakkun et al., 1993;
Miyazaki, 1991). In Sri Lanka flesh of frogs and snakes
is not eaten and any other meat (for example pork) is
fully cooked before eating; poultices made from flesh
are rare. Therefore, the only possible way humans can
get infected remains by drinking contaminated water.
In the first report (Wijesundera, 1997) the patient had
been drinking water that was not boiled, from an
unprotected well. In the second case report
(Wijesundera, 1997), the patient had a history of
swimming in a pool. It is possible that she accidentally
swallowed some water from the pool. In the present
case, the child visited her aunt's home frequently where
she drank well water that was not boiled.
The presence of the natural cestode hosts, namely dogs
and cats as pets is another contributing factor for human
infection. When the pets are not regularly dewormed,
they would be harbouring parasites–a ready source of

Review of sparganosis in Sri Lanka
33
infection. The man in the first case had two cats and a Andersen KI. 1983. Description of musculature differences in
dog as pets. Unprotected wells are another factor that spargana of Spirometra (Cestoda: Pseudophyllidea) and
tetrathyridia of Mesocestoides (Cestoda; Cyclophyllidea)
should be taken into consideration. Rain water or open
and their value in identification. J Helminth 57:331-334.
drains washing the surrounding surface layers could
bring worm eggs from the soil polluted with host faeces. Ausayakkun S, Siriprasert V et al., 1993. Ocular sparganosis in
This would contaminate the water, which when drunk Thailand. Southeast Asian J Trop Med and Pub Health
24:603-606.
without boiling can bring about the infection.
Chandler C. 1955. The Cestoidea or Tapeworms. In: Introduction
The early removal of sparganum is important
th
to Parasitology. C. Chandler (Ed.) 9 Edition. John Wiley and
considering the facts that some of the larvae could live Sons, Inc., New York. pp 329-374.
for over five years, that they have the ability to migrate
Dissanaike AS, Anthonis PR et al., 2001. Two more cases of
inside the body and to becoming invasive, and thus to
sparganosis from Sri Lanka. The Ceylon J Med Sci 44:19-22.
cause proliferative sparganosis. Additionally, the larvae
can damage tissues of vital organs leading to serious Holodniy M, Almenoff J et al., 1991. Cerebral sparganosis: case
consequences. Headache and seizures, with or without
report and review. Rev Infect Dis 13:155-159.
focal neurological signs, are common indications of Kron MA, Guderian R et al., 1991. Abdominal Sparganosis in
cerebral sparganosis (Anders et al., 1984). The remedy Ecuador: a case report. Am J Trop Med Hyg 44:146-150.
of choice that has been curative for both cerebral and Miyazaki I. 1991. Spirometriasis. In: An illustrated book of
subcutaneous sparganosis has been the surgical Helmintic Zoonoses. Miyazaki I. (Ed.) Tokyo: International
excision of the parasite (Holodniy et al., 1991).
Medical Foundation of Japan. pp 207-214.
In Sri Lanka, because raw or undercooked meat is Norman SH and Kreutner A. 1980. Sparganosis: clinical and
pathologic observation in ten cases. Southern Med J 73:297-
seldom if ever consumed and application of poultices
300.
prepared from paratenic hosts is not practiced in
traditional medicine, the simplest way to prevent Pampiglione S. And Fioravanti ML et al., 2003. Human
human sparganosis is to use boiled water for drinking sparganosis in Italy. Case report and review of the European
cases. Acta pathologica, microbiologica et immunologica
purposes.
Scandinavica 111:349-354.
ACKNOWLEDGEMENTS Rehak M, Kolarova L et al., 2006. Ocular sparganosis in the
Czech Republic–a case report. Klinick'a mikrobiologie a
We wish to thank Professor A. S. Dissanaike, Emeritus Infekc ni Le'kar stvi 155:161-164.
Professor, Department of Parasitology, Faculty of
Medicine, University of Colombo, Sri Lanka, for his Seneviratna, P. 1955. A check list of helminths in the Department
of Veterinary Pathology. University of Peradeniya. Ceylon
valuable comments and Ms. R. L. Dewasurendra of the
Vet J 3:32-37.
same Department for assistance. We acknowledge the
assistance provided by Mr. R. A. A. N. Ranawaka of the Seneviratna P and Mahalingam S. 1962. Some helminths hitherto
Audio Visual Unit, Faculty of Medicine, University of not reported from Ceylon. Ceylon Vet J 10:88-92.
Colombo and Ms. R. A. D. M. Jayanthi of the Tsou M and Huang TW. 1993. Pathology of Subcutaneous
Postgraduate Institute of Medicine, University of sparganosis: report of two cases. J Formosa Med Assoc 92:
Colombo, for microphotography.
649- 653.
REFERENCES
Wijesundera M de S, Ratnatunga N et al., 1997. First reports of
subcutaneous sparganosis in Sri Lanka. Ceylon Med J 42:30-
Alibhoy AT, Fernando R et al., 2007. A case of cerebral 32.
sparganosis. Ceylon Med J 52:93-94.
Anders K, Foley K et al., 1984. Intracranial sparganosis: an
uncommon infection. J Neurosurgery 60:1282-1286.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 34-41
Original paper
J P D
Immunocytochemical demonstration of 5-hydroxytryptamine
and localization of monoamine oxidase in
Gigantocotyle explanatum and Gastrothylax crumenifer
(Digenea: Paramphistomidae)
1 2 3
S. Ghani , Z. Ghani and S. M. A. Abidi
1
Department of Zoology, Sunbeam College for Women, Varanasi.
2
Department of Zoology, A. K. A. P. G. College, Varanasi.
3
Section of Parasitology, Department of Zoology, Aligarh Muslim University, Aligarh.
Received 13 March 2008; revised 27 May 2008; accepted 30 June 2008
ABSTRACT. The biogenic amine, 5-hydroxytryptamine (5-HT or serotonin) and its metabolizing
enzyme monoamine oxidase (MAO) were found to be differentially distributed in different regions of
liver and rumen amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer, respectively,
which infect Bubalus bubalis. Strong immunofluorescence, which indicated the presence of 5-HT,
revealed the presence of serotoninergic network in the suckers, pharynx, tegumental and subtegumental
musculature, parenchyma and uterine region of both the amphistomes. Intense
immunoreactivity in the muscular gonopore region was, however, more prominent in G. explanatum.
The luminal contents of gut in G. crumenifer also displayed strong reactivity for 5-HT, whereas
diffused distribution of this neurotransmitter was observed in the ventral pouch of the rumen
parasite. The biogenic amine catabolizing enzyme MAO was also found to be co-distributed with
differential stain intensity in various regions of the two amphistomes. Strong MAO activity was colocalized
in the oral sucker, vitellaria and subtegumental regions of both the parasites, whereas the
enzyme activity was sparsely distributed in gonads. Distinct distribution of MAO was also evident in
the acetabulum region of rumen dwelling worms, whereas in the worms of liver a comparatively weak
enzyme activity was recorded. Such variations in the distribution of 5-HT and MAO could be due to
the differences in the physicochemical nature of the microenvironment as well as feeding preferences
of the host that might exert an influence on the parasites resulting into adaptive variations exhibited
by the amphistome species which infect the same host but occupy different microenvironment.
Keywords: amphistome, histochemical localization, 5-hydroxytryptamine, immune-reactivity,
monoamine oxidase
INTRODUCTION recognized in platyhelminths, among which 5-
hydroxytryptamine (5-HT) appears to be the dominant
A number of neuronal messengers have been well
biogenic amine in all classes (Bennett et al., 1969; Lee
et al., 1978; Fairweather et al., 1987; Maule et al., 1993;
Corresponding author: Dr. S. M. A. Abidi, Section of
Parasitology, Department of Zoology,Aligarh Muslim
Halton et al., 1994; Reuter et al., 1995; Mair et al.,
University, Aligarh-202 002, India.
2000; Trim et al., 2001; Stewart et al., 2003; El-Naggar
E-mail: abbasabidi92@hotmail.com

Localization of 5-HT and MAO in amphistomes
35
et al., 2004; 2007). In helminthes, 5-HT has a number of in primary antiserum at a dilution of 1:5 in PBS for 18 h
important roles in behavioural and physiological at 4°C. After washing for 30 min in PBS, slides were
processes like feeding, locomotion, circadian rhythms, incubated with fluorescine isothyocyante (FITC)-
learning and memory, synaptic growth and plasticity labeled anti-rabbit IgG at a dilution of 1:100 for 40 min
(Walker et al., 1996). In addition to its significance in at 4°C. The sections were washed again in PBS for 90
neurotransmission, 5-HT also exerts an influence on the min, mounted in glycerol and examined under
carbohydrate metabolism, particularly on glycolysis ultraviolet light under an Olympus BH fluorescence
and glycogenolysis in parasites (Rahman and Mettrick, microscope. The control slides were processed
1 9 8 5 ) . 5 - H T e n h a n c e s t h e a c t i v i t y o f simultaneously but were not treated with primary
phosphofructokinase, phosphorylase and stimulates the antiserum.
uptake of glucose (Mansour, 1959a; 1959b; Mansour
and Mansour, 1962; Mansour, 1979; Rahman and
Histoenzymological localization of MAO: For the
Mettrick, 1985). The immunoregulatory role of 5-HT
histochemical localization of MAO in G. explanatum
has also been reported in mammalian system, wherein and G. crumenifer, cryocut sections (8 µm thick) were
this amine suppresses the antibody production (Qiu et immediately incubated in the test medium containing
al., 1996). Although the presence of 5-HT has been 4.6 mM tyramine-HCl, 0.1M Tris-HCl, nitro blue
reported in a number of parasitic helminthes; however, tetrazolium (Nitro BT) and distilled water for 5 h at
apparently, no report is available on its distribution in 37+1°C (Abidi, 1990). Controls were also run
amphistomes and almost nothing is known about the simultaneously by similarly treating the sections at
rapid inactivation of neurotransmitter like serotonin by 80°C for 1 h, or by omitting the substrate from the
catabolizing enzyme, MAO, in amphistomes.
reaction mixture.
The monoamine oxidase is known to act as a major RESULTS
scavenger of neuroamines released at the neuronal
Immunoreactivity of 5-HT: The results of the IFA test
junctions as well as for non–neurotransmitter amines.
revealed differential distribution of 5-HT in G.
Therefore, in the present study, 5-HT and MAO have
explanatum and G. crumenifer (Table I, Fig. 1–10). The
been studied immunocytochemically and
tegumental and sub-tegumental regions of both the
histoenzymologically, respectively, in the two
w o r m s d i s p l a y e d m o d e r a t e t o s t r o n g
amphistomes occupying different micro-environments
immunofluorescence (Fig. 2 and 7), which suggest the
within the same host, Bubalus bubalis.
presence of 5-HT at the host-parasite interface.
MATERIALS AND METHODS
In G. explanatum, moderate immuno-reactivity to 5-HT
Parasite collection: Mature and active flukes of revealed the aminergic network in the oral sucker and
Gigantocotyle explanatum and Gastrothylax muscular pharynx, whereas low level of biogenic amine
crumenifer were collected from the infected liver and appeared to be distributed in the parenchymatous areas,
rumen of eight Indian water buffaloes, Bubalus bubalis, thus showing weak immunofluorescence in this region
freshly slaughtered at the local abattoir. The worms (Fig. 1–5). The distribution of 5-HT appeared to be
were thoroughly washed in glucose-free Hanks' fairly strong around the gonopore, whereas low to
balanced salt saline (HBSS; pH 7.4) at 37+1°C before moderate distribution was recorded in the uterus and
being processed for various studies.
acetabular region.
Immunocytochemical localization of 5-HT: The In contrast, the distribution of 5-HT in G. crumenifer
indirect immunofluorescence antibody (IFA) technique was moderate to intense in the intestinal region,
of Coons et al. (1955) and Cable et al. (1996) was whereas the gut contents showed very strong
employed for the demonstration of 5-HT. The worms immunofluorescence. The intestinal muscles and
were embedded in Tissue-Tek II and sectioned at 10 µm
acetabular region exhibited low to moderate activity,
on a Yorco cryotome at -30 °C. The sections were
which was also evident in the ventral pouch region.
mounted on glass slides and air-dried for 1 h, followed
However, in oral sucker, pharynx, uterus and
by blocking of non-binding sites with blotto (5%
parenchymatous region, weak activity was observed
skimmed milk prepared in phosphate-buffered saline
(Fig. 6–10). Immunofluorescence was completely
(PBS) for 4 h. Thereafter, the sections were washed in
absent from the syncitial layer of the tegument in both
PBS for 10 min x 3–4 times at 25°C, and then incubated
the amphistomes under study.

36 Ghaniet al.
Fig. 1–5. Immunocytochemical localization of 5-HT in different regions of liver amphistome, G. explanatum.
Os: oral sucker, Sc: syncitial layer, Stg: sub-tegument, Pa: parenchyma, Go: gonopore, Ph: pharynx, Eg: egg, Ac: acetabulum.
Scale bar = 8 m, magnification x 100
Fig. 6–10. Immunocytochemical localization of 5-HT in different regions of rumen amphistome, G. crumenifer.
Os: oral sucker, Nc: nerve cord, Sc: syncitial layer, Stg: sub-tegument, Pa: parenchyma, Go: gonopore, Ph: pharynx, Eg: egg,
Ic: intestinal caeca Ac: acetabulum, Vp: ventral pouch.
Scale bar = 8 m (Fig. 6, 7, 9), Scale bar = 28 m (Fig. 8, 10) Magnification= Fig. 6, 7, 9 x1 00, Fig. 3, 5 x 200

Localization of 5-HT and MAO in amphistomes
37
Fig. 11–18. Histoenzymological localization of monoamine oxidase in different regions of G. explanatum and G. crumenifer.
Os: Oral sucker, Tg: Tegument, Stg: Sub-tegument, Pa: Parenchyma, Vit: Vitellaria, Ic: Intestinal caecae, Im: Intestinal
muscles, Il: Intestinal lumen, Ts: testes, Ov: ovary, Es: egg shell, Vp: ventral pouch, Cm: circular muscles, Ac: acetabulum,
Acm: acetabular muscles. Scale bar = 5 m.(Fig. 11–18)
Magnification = x 100

38 Ghaniet al.
Table I. Immunocytochemical localization of 5-HT in G. explanatum and G. crumenifer
5-HT immunoreactivity in
Worm region G. explanatum G. crumenifer
Syncitium - -
Tegument + + + + + +
Sub- tegument + + / + + + + +
Oral sucker + + +
Pharynx + + +
Intestinal caecae
(i) Intestinal muscles - + / + +
(ii) Intestinal lumen - ++ / + ++
Parenchyma + +
Ventral pouch + +
Gonads
(i) Testis - -
(ii) Ovary - -
Gonopore + ++
Uterus +/ + + +
Acetabulum
(i) Outer lining + / + + + / + +
(ii) Inner lining + / + + + / + +
- absent; + low; + + moderate; + + + intense; + / + + low to moderate; + + / + + + moderate to intense
Table II. Histoenzymological localization of MAO in liver and rumen amphistomes, G. explanatum and G.
crumenifer
Enzyme activity in
Worm region G. explanatum G. crumenifer
Syncitium - -
Sub-tegument + + / + + + + + / + + +
Oral sucker + + + + / + + +
Intestinal caecae
(i) Intestinal muscles + + +
(ii) Intestinal lumen - -
Vitellaria + + + + +
Parenchyma + +
Ventral pouch + + +
Gonads
(i) Testes + + / + + + + + / + + +
(ii) Ovary - / + - / +
Acetabulum
(i) Outer periphery + +
(ii) Middle + + + / + + +
(iii) Inner periphery + +
- absent; + low; + + moderate; + + + intense; - / + absent to low; ++ / + + + moderate to intense
MAO localization: MAO was found to be trematode, whereas intense activity was observed in
differentially distributed, as revealed by the formazan vitellaria. The oral sucker and intestinal muscles
deposits, but appeared to be substantially co-localized displayed moderate activity, but weak enzyme activity
in the areas where 5-HT was also found to be distributed was recorded in the parenchymatous and the acetabular
in both the parasites under study (Table II, Fig. 11–18). regions. The MAO activity was indistinct in the ovary
Moderate to intense enzyme activity was recorded in and was totally absent from the intestinal lumen (Fig.
the tegument, sub-tegument and testes of the liver 11–14).

Localization of 5-HT and MAO in amphistomes
39
The sub-tegumental, oral sucker and testes regions of feeding because in C. elegans the occurrence of 5-HT
G. crumenifer displayed moderate to strong enzyme was found to be necessary for stimulating the
activity. The localization of MAO in the ventral pouch pharyngeal pumping (Raizen and Avery, 1994; Niacaris
region was also strong. The enzyme activity was and Avery, 2003), and the process of ingestion could be
sparsely distributed in the surrounding tissues of the blocked by the serotonin antagonist, gramine (Avery
intestinal caecae. The characteristic distribution pattern and Horvitz, 1990). Similarly, the distribution of 5-HT
of MAO could be seen within the acetabular region of has also been demonstrated in the pharyngeal region of
this trematode (Fig. 15–18). other helminthes, G. vitelliloba, F. hepatica and in A.
DISCUSSION
suum (McKay et al., 1991; Brownlee et al., 1994; 1995;
Gustafsson et al., 2001), indicating its role in
The present investigation revealed differential neuromuscular coordination.
distribution of serotoninergic network in G.
However, a marked difference in the distribution of 5-
explanatum and G. crumenifer. The differential
HT in the intestinal caecae of liver and rumen
immunofluorescence for 5-HT was observed in both the
amphistomes was observed. It was totally absent in the
worms, where tegumental and sub-tegumental regions
intestinal caeca of liver amphistome, whereas moderate
displayed moderate to intense immunoreactivity. It is
to intense immunoreactivity was present in the outer
well known that 5-HT plays an important role in
periphery and intestinal lumen of the rumen parasite,
neuromuscular coordination, thus, indicating its
but MAO activity was observed in the sub-mucosal and
potential role in the motility of the two amphistomes as
intestinal muscles of both the parasites. Since about
also reported for other digenetic trematodes, S.
90% of the total body content of 5-HT is found within
mansoni, F. hepatica, B. gracilescens and the
the gastrointestinal tract (Erspamer, 1966), therefore, it
nematodes, A. tetraptera and A. suum (Anya, 1973;
is possible that 5-HT of host origin may also be
Fairweather et al., 1984; 1987; Mair et al., 2000; Trim
absorbed via the alimentary route in G. crumenifer, as
et al., 2001; Stewart et al., 2003). These results were
suggested by Martin and Donahue (1989) for A. suum
further augmented by the co-distribution of strong
that 5-HT can be absorbed by the gastrointestinal
MAO activity in sub-tegumental region. Adults and
epithelial cells, therefore, the presence of strong
developmental stages of parasitic flatworms rely on
immunofluorescence for 5-HT in the lumen of rumen
well-developed neuromuscular system for host
amphistome is not surprising. However, the distribution
a t t a c h m e n t . T h e o c c u r r e n c e o f s t r o n g
of MAO in close association with gastrointestinal tract
immunoreactivity for 5-HT in the selective regions of
could provide a detoxification mechanism to protect the
oral sucker revealed a rich supply of serotoninergic
parasite from any deleterious effects of excess amount
fibres in the well developed muscular organ of the
of biogenic amines of host origin.
amphistomes. However, 5-HT was sparsely distributed
in the acetabular region, whereas strong MAO activity The presence of 5-HT and MAO in the parenchymal
could be localized in the peripheral region, close to the region of both the amphistomes and particularly in the
attachment surface area. It has been suggested that muscular lining of ventral pouch of G. crumenifer could
MAO could also participate at the host-parasite be due to the presence of mitochondria, which are
interface in the inactivation of exogenous amines which known to occur along the lining of ventral pouch and in
come from dietary intake by the host (Barrett, 1981). the parenchymal tissues.
Moreno and Barrett (1979) suggested that in H.
diminuta the main function of monoamine oxidase is to
The serotoninergic component of neuromuscular
protect the parasite against exogenous amines present
coordination has been reported in the reproductive
in the host alimentary tract, where bacteria also
system of F. hepatica, Moniezia expansa, D. sagittata,
synthesize biogenic amines (Nag et al., 1978).
and H. medioplexus by Fairweather et al., 1987; Maule
Therefore, MAO can be considered as an important
et al., 1993; Cable et al., 1996; Humphries et al., 1997,
component at the interface detoxifying the excess
respectively. Similarly, the strong immunoreactivity for
amines in order to maintain a successful host-parasite
5-HT in the gonopore region of G. explanatum and in
relationship.
the uterine musculature of both the flukes suggests the
role of 5-HT in reproductive physiology of the
The 5-HT immunoreactivity in the pharynx of both the amphistomes, where 5-HT, beside other putative neuroamphistomes
indicate serotoninergic regulation of transmitters, might be partially involved in vigorous
pharyngeal musculature, thus suggesting its role in contractions of uterine wall during the shaping and

40 Ghaniet al.
passage of eggs, but the higher activity in G. Anya AO. 1973. The distribution and possible
explanatum could possibly be due to the fact that neurophamacological significance of serotonin (5-
hydroxytryptamine) in Aspicularis tetraptera (Nematoda).
enormous egg production continues through out the
Comp Gen Pharmacol 4:149-156.
year in this parasite, whereas strict seasonal
reproductive cycle is operative in rumen amphistome, Avery L and Horvitz HR. 1990. Effects of starvation and
G. crumenifer (Hanna et al.,1988). Therefore, it could neuroactive drugs on feeding in Caenorhabditis elegans. J
Exp Zool 253:263-270.
be possible that any chemotherapeutic intervention in
the metabolism of 5-HT could influence the egg Barrett J. 1981. Biochemistry of Parasitic Helminthes. London
producing activity of these parasites. The presence of and Basingstoke, Macmillan publishers Ltd.
monoamine oxidase in gonads is consistent with the Bennett JL, Bueding E, Timms AR and Engstrom RG. 1969.
known transmitter and hormonal role of 5-HT in Occurrence and levels of 5-hydroxytryptamine in
reproductive activity of trematodes. The MAO activity Schistosoma mansoni. Mole Pharmacol 5:542-545.
has also been reported in the gonads of female A. galli
Brownlee DJA, Fairweather I, Johnston CF and Shaw C. 1994.
and S. cervi (Mishra et al., 1983; Agarwal et al., 1990). Immunocytochemical demonstration of peptidergic and
In the present study the absence of fluorescence for 5- serotoninergic components in the enteric nervous system of
HT in the ovary and testes but the presence of MAO in the round worm Ascaris suum (Nematoda; Ascaroidea).
these regions is of some curiosity. It is possible that this Parasitol 108:89-103.
biogenic amine could be present in the gonads but the Brownlee DJA, Holden-Dye L, Fairweather I and Walker RJ.
concentration may be too low to be detected by the 1995. The action of serotonin and the nematode neuropeptide
methods used in the present study. The KSAYMRF amide on the pharyngeal muscle of the parasitic
immunoreactivity in the eggshells of only G. nematode, Ascaris suum. Parasitol 3:379-384.
explanatum could possibly be due to auto-fluorescence, Cable J, Marks NJ, Halton DW, Shaw C, Johnston CF, Tinsley
which was totally absent in case of G. crumenifer.
RC and Gannicott AM. 1996. Cholinergic, serotoninergic and
peptidergic components of the nervous system of Discocotyle
It is concluded that 5-HT, in concurrence with MAO, is sagittata (Monogenea: polyopisthocotylea). International
widely distributed in amphistomes but the distribution Journal for Parasitol 26:1357-1367.
varies in different regions with some specific
Coons AH, Leduc EH and Connolly JM. 1955. Studies on
differences in parasites occupying different antibody production: I. A method for the histochemical
microenvironments. The pronounced localization of demonstration of specific antibody and its application to a
MAO in the sub-tegument, particularly in the study of the hyperimmune rabbit. J Exp Med 102:49-60.
acetabular region of the rumen amphistome suggests
El-Naggar MM, Arafa SZ, El-Abbassy SA, Stewart MT and
the involvement of this enzyme at the interface for the Halton DW. 2004. Neuromusculature of Macrogyrodactylus
establishment of host-parasite relationship.
clarii, a monogenean gill parasite of the Nile cat fish Clarias
ACKNOWLEDGEMENTS
gariepinus in Egypt. Parasitol Res 94:163-175.
El-Naggar MM, Arafa SZ, El-Abbassy SA, Stewart MT and
The authors are thankful to the Chairman, Department Halton DW. 2007. Neuromusculature of Macrogyrodactylus
of Zoology, Aligarh Muslim University, Aligarh, and congolensis, a monogenean skin parasite of the Nile cat fish
Clarias gariepinus. Parasitol Res100:265-279.
Dr. Haris M. Khan of J. N. Medical College, Aligarh,
for providing the necessary laboratory facilities, and to Erspamer V. 1966. Occurrence of indole alkylamines in nature.
Dr. M. Khalid Saifullah for his comments and Mr. In: Handbook of Experimental Pharmacology. Erspamer V.
Ramesh Chandra for parasite collection. Financial (Ed.), Springer, Berlin, Heidelberg, New York. pp 132-181.
Assistance from University Grants Commission, New Fairweather I, Holmes SD and Threadgold LT. 1984. Fasciola
Delhi, and Council of Science and Technology, U. P, hepatica motility response to fasciolicides in vitro. Exp
Lucknow, is gratefully acknowledged.
Parasitol 57:209-224.
REFERENCES
Fairweather I, Maule AG, Mitchell SH and Halton DW. 1987.
I m m u n o c y t o c h e m i c a l d e m o n s t r a t i o n o f 5 -
Abidi SMA. 1990. Physiological studies of some gastrointestinal hydroxytryptamine (serotonin) in the nervous system of the
helminthes of ruminants. Ph.D. Thesis, Aligarh Muslim liver fluke, Fasciola hepatica (Trematoda, Digenea).
University. Aligarh, India.
Parasitol Res73:255-258.
Agarwal A, Shukla OP, Ghatak S and Tekwani BL. 1990. Gustafsson MKS, Terenina NB, Kreshchenko ND, Reuter M,
Biogenic amines metabolites and monoamine oxidase in the Maule AG and Halton DW. 2001. Comparative study of the
filarial worm Setaria cervi. Int J Parasitol 20:873-881.
spatial relationship between nicotinamide adenine

Localization of 5-HT and MAO in amphistomes
41
dinucleotide phosphate-diaphorase activity, serotonin Maule AG, Halton DW, Shaw C and Johnston CF. 1993. The
immunoreactivity and GYIRF amide immunoreactivity and cholinergic, serotoninergic and peptidergic components of
the musculature of the adult liver fluke, Fasciola hepatica the nervous system of Moniezia expansa (Cestoda,
(Digenea, Fasciolidae). J Comp Neurol 429:71-79. Cyclophyllidea). Parasitol 106:429-440.
Halton DW, Maule AG, Brennan GP, Shaw C, Stoitsova SR and McKay DM, Halton DW, Johnston CF, Fairweather I and Shaw
Johnston CF. 1994. Grillotia erinaceus (Cestoda, C. 1991. Cytochemical demonstration of cholinergic,
Trypanorhyncha): localization of neuroactive substances in serotoninergic and peptidergic nerve elements in
the plerocercoid, using confocal and electron microscopic Gorgoderina vitelliloba (Trematoda: Digenea). Int J Parasitol
immunocytochemistry. Exp Parasitol 79:410-413. 21:71-80.
Hanna REB, Williamson DS, Mattison RG and Nizami WA. Mishra SK, Sen R and Ghatak S. 1983. Monoamine oxidase in
1988. Seasonal reproduction in Paramphistomum epiclitum adult Ascaridia galli. J Helminthol 57:313-318.
and Gastrothylax crumenifer, rumen paramphistomes of the
Indian water buffalo and comparison with the biliary
Moreno MS and Barrett J. 1979. Monoamine oxidase in adult
paramphistome Gigantocotyl explanatum. Inte J Parasitol
Hymenolepis diminuta (Cestoda). Parasitol 78:1-5.
18:513-521. Nag M, Kalyan C and Nandi N. 1978. Monoamine oxidase and
Humphries JE, Halton DW, Johnston RN, Maule AG, Johnston
amine tetrazolium reductase in the guinea pig small intestine.
CF and Shaw C. 1997. Cholinergic, serotoninergic and
Ind J Exp Biol 16:174-176.
peptidergic components of the nervous system of Niacaris T and Avery L. 2003. Serotonin regulates repolarization
Haematoloechus medioplexus (Trematoda, Digenea), of the C. elegans pharyngeal muscle. J Exp Biol 206:223-231.
Characterised by cytochemistry. Int J Parasitol 27:517-525.
Qiu Y, Pang Y and Wang J. 1996. Immunoregulatory role of
Lee MB, Bueding E and Schiller EL. 1978. The occurrence and neurotransmitters. Adv Neuroimmunol 6:223-231.
distribution of 5-hydroxytryptamine in Hymenolepis
diminuta and Hymenolepis nana. J Parasitol 64:257-264. Rahman MS and Mettrick DF. 1985. Schistosoma mansoni,
effects of in vitro serotonin (5-HT) on aerobic and anaerobic
Mair GR, Maule AG, Day TA and Halton DW. 2000. A confocal carbohydrate metabolism. Exp Parasitol 60:10-17.
microscopical study of the musculature of adult Schistosoma
mansoni. Parasitol 121:163-170.
Raizen DM and Avery L. 1994. Electrical activity and behaviour
in the pharynx of Caenorhabditis elegans. Neuron 12:483-
Mansour TE. 1959a. The effect of serotonin and related 495.
compounds on the carbohydrate metabolism of the liver fluke
Fasciola hepatica. J Pharmacol Exp Ther 126:212-216.
Reuter M, Gustafsson MKS, Maule AG and Shaw C. 1995. The
nervous system of Tricladida. II Neuroanatomy of Dugesia
Mansour TE. 1959b. Studies on the carbohydrate metabolism of tigrina (Paludicola, Dugesiidae): an immunocytochemical
the liver fluke, Fasciola hepatica. Biochem Biophys Acta study. Invert Neurosci1:133-143.
34:456-464.
Stewart MT, Marks NJ and Halton DW. 2003. Neuroactive
Mansour TE. 1979. Chemotherapy of parasitic worms: new substances and associated major muscle systems in
biological strategies. Science. 205:462-469.
Bucephaloides gracilescens (Trematoda:Digenea)
Mansour TE and Mansour JM. 1962. Effects of serotonin (5-
metacercaria and adult. Parasitol Res 91:12-21.
hydroxytryptamine) and adenosine 3',5'-phosphate on Trim JE, Holden-Dye L, Willson J, Lockyer M and Walker RJ.
phosphofructo-kinase from the liver fluke, Fasciola 2001. Characterization of 5-HT receptors in the parasitic
hepatica. J Biol Chem 237:629-634. nematode, Ascaris suum. Parasit 122:207-217.
Martin RE and Donahue MJ. 1989. Tissue and ultrastructural
localization of 5-hydroxytryptamine (serotonin) in the tissues
of Ascaris suum with energy dispersive X-ray spectrometry
of immunoreactive structure. Int J Parasitol 19:585-596.
Walker RJ, Brooks HL and Holden-Dye. 1996. Evolution and
overview of classical transmitter molecules and their receptor
in Halton, DW & Chappell, LH (Eds.) Molecular
Biochemistry and Physiology. Parasitology 113: S3-S33.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 42-46
Original paper
Seasonal dynamics in lungworm infections in sheep
J P D
N. Moghaddar, S. S. Shekarforoush and A. Afrahi
School of Veterinary Medicine, Shiraz University, Shiraz, Islamic Republic of Iran
Received 29 January 2008; revised 31 March 2008; re-revised 26 May 2008; accepted 2 August 2008
ABSTRACT. Out of 1804 lungs of sheep examined for lungworms during 2001-2005 in abattoirs of
Shiraz, Iran, 529 (29.3 %) of them were found positive. Majority (67.0%) of the lungs had mixed
infection with more than one lungworm species. Seasonal analysis of the occurrence of infection
revealed a significantly high (p > 0.1) incidence in spring (38.5 %) and summer (34.5 %), followed
by autumn (16.7%) and winter (8 %). Muellerius capillaries (51.46 %) and Dictyocaulus filaria
(31.58%) were the predominant worm species, whereas Protostrongylus rufescens (12.54%) and
Cystocaulus ocreatus (4.42%) were relatively less common. Coprological examination
demonstrated a significantly high (p > 0.1) incidence of infection in sheep kept by tribal people
(nomadic people) than those by local ones, as shown by higher intensity of infection and clinical
signs in the tribal animals. Bronchitis, pneumonia with intense damage of parenchyma, and
presence of a large number of nodules of variable size resulted in the rejection of infected lungs.
Effective control measures are suggested.
Keywords: lungworms, Iran, seasonal dynamics, sheep, tribal people
INTRODUCTION
MATERIALS AND METHODS
All over the world, lungworm infections in sheep have Out of a total 2224 sheep included in this study, 978
been well recognized (Rose, 1965; Sammarrae and belonged to the local farmers around Shiraz, the
Sewell, 1977; Dhar, 1985), and have also been capital of Fars Province of Iran, and 1246 to the tribal
reported from Iran (Eslami et al., 1980; Moghaddar et people. The observations were recorded in all seasons
al., 1991). However, apparently, no systematic studies throughout the year, depending upon the availability
on seasonal incidence of lungworm infections in of animals. The tribal people raised and purchased the
Iranian sheep have so far been made. The present animals from different agro-climatic zones, and
investigations were, therefore, aimed to study the moved with them in spring and summer in search of
seasonal dynamics of the infections caused by grazing places and suitable markets for sale. The
different lungworms species and their clinico animals were assigned to two groups viz. Group-I:
pathological implications in sheep of local farmers twenty sheep belonging to tribal people in each
and tribal people (nomadic people) in Fars Province of season except in winter (total 180) and twenty from
Iran.
local farmers in each season (total 240), which
showed respiratory distress were selected during
2003-2005 for clinical and coprological examinations
and the detailed clinical examination of the selected
animals was done afterwards, Group-II: local
Corresponding author: Dr. N. Moghaddar, School of Veterinary animals and those belonging to tribal people (total
Medicine, Shiraz University, Shiraz, Islamic Republic of Iran.
1804) were also examined in abattoirs of Shiraz during
E-mail: nasrinmoghaddar@yahoo.com
2001-2005.

Lungworm infections in sheep
43
For coprological examination, the faecal samples significantly (p > 0.1) high incidence was observed in
from individual animal were collected per rectum in spring (38.5 %) and summer (34.5 %), as compared to
suitable containers, and examined afresh as far as autumn (16.7 %) and winter (8.0%). Majority (57.6 %)
possible. Of these, a few positive samples were used of the specimens showed low grade infection, whereas
for the culture of larvae. The samples collected from 67.0 % had mixed infection with more than one
remote areas were preserved in 10% buffered species. The species recovered were Muellerius
formalin, and then taken to the laboratory. Faecal capillaries (51.46%), Dictyocaulus filaria (31.58%),
examination was performed by direct smear method, Protostrongylus rufescens (12.54%) and Cystocaulus
and the faecal cultures were raised from fresh, positive ocreatus (4.42%), but M. capillaris and D. filaria
faecal samples in petri plates/glass jars depending predominated in both local animals and animals of
upon the faecal quantity (Soulsby, 1982). The tribal people. The faecal examination revealed (Table
lungworms' larvae from faecal cultures were collected II; Fig. 2) 31.7% and 30.0 % infection during spring
by Baermann technique (Soulsby, loc.cit). Necropsy and summer seasons, respectively, followed by
examination in abattoirs was carried out by examining 12.5% and 11.6 % infection in autumn and winter,
the intact lungs for gross lesions and after incision for respectively. Animals, which belonged to tribal people
lesions in parenchyma and bronchi. The worms were showed significantly (p > 0.1) high infection as
collected from different locations of lungs, washed in compared to the local ones; however, the former could
physiological saline and collected in tubes. Pieces of not be examined during winter due to their nonlungs
were also used in Baermann apparatus to isolate availability.
the lungworms. Alternatively, the lung pieces were
Gross examination of the lungs showed variable
also placed in warm saline and isolated by
lesions depending upon the worms' species involved,
sedimentation and slow centrifugation. The
their number and the duration of infection. Severely
worms/larval stages thus recovered were counted by
affected lungs were highly congested and enlarged. At
dilution technique. The worms and the juveniles were
places, the organs were hard to touch, while at other
fixed and preserved in 5 or 10 % formol saline for
places showed firm, raised dark grayish or palefurther
processing and identification. The data were
yellow nodules of different size. On incision, the
analysed statistically by Chi-square test of
bronchi contained worms and large amount of mucus
significance.
and exudate mixed with blood. Adult D. filaria and P.
RESULTS rufescens worms were observed to block air
passages. The nodules when present, were
Out of 1804 sheep lung specimens examined, 529
circumscribed or enlarged, embedded in the lung
(29.3%) were positive with incidence ranging from
parenchyma and contained spirally coiled adult M.
5.7% to 48.0 % in different seasons (Table I; Fig. 1). A
Table I. Seasonal incidence of lungworms of sheep recorded in abattoirs of Shiraz, Iran
Year of
Seasons of the year
examination Spring Summer Autmn Winter
T +ve %ve T +ve %ve T +ve %ve T +ve %ve
2001 75 36 48.0 222 84 37.8 60 19 31.6 45 3 6.6
2002 95 31 32.6 146 44 30.1 66 8 12.1 25 2 8.0
2003 101 42 41.5 155 49 31.6 75 13 17.3 45 4 8.9
2004 93 25 26.8 120 53 44.1 73 9 12.3 35 2 5.7
2005 85 39 45.8 135 39 28.9 79 20 25.3 74 7 9.4
Total 449 173 38.5* 778 269 34.5* 353 69 16.7 224 18 8.0
Grand total 1804 529 29.3 %
*Significant difference: (p > 0.1) ; T = number of animals examined ; +ve = positive ;
% ve = percent positive.

44 Moghaddar et al.
Table II. Seasonal incidence of lungworms in sheep by faecal examination in Shiraz, Iran
Animals /
Year of
Seasons of the year
Examination Spring Summer Autumn Winter
+ve % +ve % +ve % +ve %
Tribal animals
2003 8 - 6 - 4 - N -
2004 10 - 11 - 3 - N -
2005 9 - 12 - 4 - N -
Total 27 45.0* 29 48.3* 11 18.3 0.0 0.0
_________ ______________ ___________ _____________
Local animals
2003 2 - 3 - 2 - 1 -
2004 5 - 2 - 0 - 3 -
2005 4 - 2 - 2 - 3 -
Total 11 18.3 7 11.6 4 6.6 7 11.6
___________ ______________ ______________ _____________
Grand total (120) 38 31.7* (120) 36 30.0* (120)15 12.5 (60)7 11.6
No. of sheep examined in each season = 20
*Significant difference (p > 0.1) ; + ve = animals positive ; % = percent ; N = nil (not examined);
Figures in parenthesis indicate total no. of animals examined.
capillaries. On few occasions, C. ocreatus (4.42%)
were also recovered. In moderate infections (50-100
worms), the organs were less affected, whereas the
60
50
low-grade infection (less than 50 worms) showed no
perceptible characteristic lesions in the lungs.
Periodic clinical examination of local and tribal's
2001
2002
2003
Percent positive
40
30
20
38.5
34.6
19.5
2004
2005
Total
10
8
0
Spring Summer Autumn Winter
Seasons of the year
Fig. 1. Seasonal incidence of lungworms of sheep recorded in abattoirs of Shiraz, Iran.

Lungworm infections in sheep
45
Percent positive
100
90
80
70
60
50
40
30
20
10
0
Spring Summer
2003
2004
Seasons of year
Tribal's sheep
2005
Local sheep
Autumn Winter Spring Summer AutumnWinter Spring Summer Autumn Winter
Fig. 2. Seasonal incidence of lungworms in tribal's and local sheep by faecal examination in Shiraz, Iran.
sheep infected with lungworms, showed variable and goats in Himalayan region of India. Except for
symptoms depending upon the intensity of infection. few sporadic reports on the occurrence of lungworms
The animals in carrier stage exhibited no marked in sheep in certain parts of Iran, no efforts were yet
symptoms except for slight nasal discharge. In made to investigate the incidence and other aspects of
moderate infections, the symptoms were nasal these important parasites particularly in the animals
discharges, coughing, dyspnea and bronchitis. of tribal people though these animals are a major
However, in heavy infections, the animals showed source of mutton in this region. The present
profuse nasal discharges mixed with blood and mucus, investigations, carried out for five years for the first
uncontrolled coughing, dyspnea, emaciation, time in this country, envisaged the need to evolve and
bronchitis and pneumonia. Few animals showed implement suitable control measures against these
raised body temperature.
parasites, and to study the epidemiology of this
DISCUSSION
infection in animals belonging to tribal people.
The observations on the gross lesions caused by
The present study established the endemicity of Fars
lungworm species in sheep, in the course of present
Province of Iran for lungworm infections. The
study, corroborated with the earlier reports of
incidence was significantly (p > 0.1) high during
Moghaddar et al. (1991 ) and those of Nimmo (1979),
spring and summer seasons in all categories of the
Pybus and Shave (1984), and Oryan and Moghaddar
animals but the animals of tribal people showed
(1991). However, the association of P. rufescens and
comparatively higher rate of infection. These results
C. ocreatus with the observed extra-nodular lung
were more or less in agreement with the solitary report
lesions was interesting.
of Moghaddar et al. (1991) who observed a quite high
(52.46%) incidence of this infection in sheep during ACKNOWLEDGEMENT
six months of their study between April and September
The authors thank the Vice-chancellor, Research for
in Shiraz area. Earlier reports of Eslami and
financial support.
Monasheri (1978) and Eslami et al. (1980) on the
prevalence of lungworms in sheep from Tehran and REFERENCES
some other adjoining areas also confirmed their
Dhar D N. 1985. Bovine and ovine parasitic bronchitis with
existence in the country. The high incidence in animals particular reference to the work done in India. In
of tribal people in the present study was possibly the “Perspectives in Parasitology”. 1:1-88.
result of frequent movements of sheep in different
Eslami A and Monasheri J.1978. Incidence of parasitic
climatic and socio-economic zones. Jithendran (1998)
infestation in sheep in Golpayigan, J Vet Facult Univ
also mentioned about the influence of migratory Tehran, Iran. 30:21-13.
grazing in the epidemiology of helminthes of sheep

46 Moghaddar et al.
Eslami A, Nikbine S and Rahbari S. 1980. Lungworms Pybus MJ and Shave H. 1984. Muellerius capillaries (Mueller,
infestation of wild sheep (Ovis ammonorientalis ) and 1889) (Nematoda: Protostrongylidae): An unusal finding in
gazella (Gazella subgutturosa ). J Vet Facult Univ Tehran, Rocky mountain big horn sheep (Ovis canadensis
Iran. 35:11-16. canadensis shaw) in South Dakota. J. Wild Life Dis 20:284-
Jithendran K P .1998. Epidemiology of gastrointestinal
288.
parasites in migratory sheep and goats in north-west humid Rose J H. 1965. The lungworms recently recorded from British
Himalayan region. Ind J Anim Sci 68:894-896. sheep. Res Vet Sci 6:189.
Moghaddar N, Oryan A and Ebrahimi M. 1991. Prevalence of Sammarrae SAA and Sewell MM H. 1977. Studies on the
lungworm infection in local breeds of sheep in Shiraz (Iran). epidemiology of Dictyocaulus filaria infection in black face
J Vet Parasitol 5:96-101. sheep on a low ground Scottish farm. Res Vet Sci 23:336-
Nimmo J S. 1979. Six cases of Verminous pneumonia
339.
(Muellerius sp. ) in goats. Canadian Vet J 20:49-50.
Soulsby E J L. 1982. Helminths, Arthropods and Protozoa of
Domesticated Animals. VII (Ed.), The English Language
Oryan A and Moghaddar N. 1991. Pathology of lungworm
Book Society and Bailliere Tindall, London. pp 773-774.
disease of sheep associated with Muellerius capillaries
infection. J Vet Parasitol 5:102-107.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 47-55
Original paper
J P D
Focus on pathogenic trematode cercariae infecting fresh
water snails (Mollusca: Gastrapoda ) of tribal region of
southern Rajasthan, India
S. L. Choubisa
Department of Zoology, Government Post Graduation College, Sirohi.
Received 12 November 2007; accepted 5 September 2008
ABSTRACT. More than 15,000 freshwater snail species, which belonged to Lymnaeidae,
Planorbidae, Viviparidae and Thiaridae families of the order Mollusca were examined for the
presence of pathogenic trematode cercariae infection. Lentic (stagnate waters) and lotic (running
waters) habitats and dwelling behaviours of these snails determined their propensity for cercariae
infection. In the lentic water, the infection was maximum in the surface dwellers and minimum in the
bottom dwellers. In the lotic habitats, in bottom dwelling snails the cercarial infection was nominal.
From these snail species, a rich diversity of cercariae was observed and six different types of cercariae
viz. amphistome, echinostome, furcocercous, gymnocephalous, monostome and xiphidiocercariae
were found. Few snail species also revealed infection with metacercariae of Strigied, Ophisthorchiide
and Aspidogaster species. The most favorable season for cercarial infection was found in the late rainy
or pre-winter season, where > 95% mature snails released different kinds of cercariae . In the morning
times, maximum number of cercariae emerged out from their snail hosts. These cercariae larvae,
having pigmented eyes on their body, are very much sensitive to light and are active swimmers. A
strong host-specificity was observed in echinostome, gymnocephalous, furcocercous and monostome
cercariae. In the present communication, the diversity of snails of lentic and lotic habitats, and their
pathogenic trematode larvae (cercariae), seasonal occurrence, behaviours and host-specificity of
various types of cercariae are discussed.
Keywords: cercaria, lentic and lotic habitats, pathogen, Rajasthan, snail hosts, trematodiasis
INTRODUCTION
The digentic trematode cercaria larva is an infective and
active stage of the development process of trematode
parasites of man and animals (vertebrates), and these
larvae are also very much pathogenic to their snail hosts
(Erasmus, 1972; Cheng, 1973; Choubisa, 1986a).
Additionally, snails are ideal bio-indicators not only for
paleo-environments and water quality (Harman, 1974;
Corresponding author: Dr. S. L. Choubisa, 205, Chanakyapuri,
Sector-4, Hiran Magri, Udaipur -313002.
E-mail: choubisasl@yahoo.com
Clarke, 1979), but for lotic and lentic aquatic
ecosystems as well (Choubisa, 1992). Hence, many
workers have reported the diversity of the snails of
lentic habitats and their pathogenic cercarial fauna from
different geographical areas (Pandey and Agrawal,
1978; Choubisa and Sharma, 1983). From Rajasthan,
only a few studies have been conducted (Choubisa,
1986b, 1991), although southern Rajasthan has a
number of perennial lentic and lotic habitats. Therefore,
the present study was undertaken to find out the
diversity of snail hosts inhabiting lentic and lotic
habitats of southern Rajasthan as well as their

48
Choubisa
pathogenic cercariae, besides the study of cercarial
behaviours, host-specificity and their seasonality.
MATERIAL AND METHODS
More than 15,000 live adult snails (gastropods), which
inhabited perennial lentic and lotic habitats of tribal
region (Banswara, Dungarpur, Chittorgarh, Rajsamand
and Udaipur districts) of southern Rajasthan, were
collected and were maintained separately in laboratory
aquaria according to their species and habitats. These
snails were identified according to Ray and Mukherjee
(1963) and Tonapi (1980). The snails were exposed to
light for the determination of cercariae infection, and
were dissected out to establish the presence of
trematode larvae. The larval trematodes were collected
and examined according to the methods reported by
Mukharjee (1980). Thus, the larvae were studied under
light cover-slip pressure, in both alive and stained
mounts. Neutral red and methylene blue stains were
used for the detailed study of penetration glands, gut
and genital rudiments. Unstained live cercariae were
studied specially for their flame cell formulae. They
were then identified following the key as given by
Erasmus (1972).
For the oberservation of behaviours and swimming
activity of naturally emerging cercariae, infected snails
were kept in separate beaker (500 ml) containing tap
water. For phototactic study, beaker containing infected
snails was covered with a black paper having few small
apertures at mid level of beaker, and then electric bulb
light was passed through these apertures. This
experiment was done in morning in a dark-room.
Simultaneously, encytorment, geotaxism and
swimming activity of different cercariae were also
observed. The thigmotaxism behaviour was observed
by contact using a needle.
RESULTS
Diversity of snails and their trematode larvae: From
the perennial lentic and lotic habitats of tribal region of
southern Rajasthan, 15 species of snails (Palte 1,
Fig.1–14) were recovered. Of these, seven species
belonged to Lymnaeidae, two to Planorbidae, two to
viviparidae, and four to Thiaridae (Mellaniidae) family
(Table I). Except the snails of Lymnaeidae family,
others were found to be bottom dwellers. From these
snail species, only one species, Malania (Plotia) scabra
was found to be stenotopic and restricted to the running
waters only.
From these snail species, basically, six types of
Plate 1, Fig. 1–14.
1. Lymnaea acuminata f. patuta
2. L. acuminata f. chlamys
3. L. acuminata f. typical
4. L. luteola f. asutralis
5. L. luteola f. typical
6. L. luteola f. impure
7. L. luteola f. rufescens
8. Indoplanorbis exustus
9. Faunus ater
10. Melania (Plotia) scabra
11. Thiara (Tarebia) lineata
12. M. striatella tuberculata
13. Vivipara bengalensis race gigantea and
14. V. bengalensis race mandiensis
cercarial larvae viz. amphistome, echinostome,
furcoercous, gymnocephalous, monostome and
xiphidocercariae were recovered. Those snail species
having surface dwelling habit (genera of Lymnaeidae
family), showed maximum diversity of trematode
cercarial larvae.

Pathogenic trematode cercariae infecting freshwater snails in Rajasthan
49
Vivipara bengalensis
Cercaria beaveri
C. komiyai
C. thapari
C. pyriformis
C. milleri
C. sewelli C. udaipuriensis
Fig. 15. Different cercarial species which emerged from Vivipara snail species.
However, metacercarial larvae such as plagiorchid and
echinostome from Vivipara bengalensis, strigeid
(Tetracotyle lymnaei) from Lymnaea acuminata
species, aspidogaster from Melania striatella
tuberculata, and opsthorchiid from Indoplanorbis
exustus snail hosts were also traced out. Different types
and species of cercariae and metacercariae from a
common snail species have been shown in Fig. 15–19.
At the same time, two different types of cercariae
(double infection) in a single snail specimen have also
been observed. In general, xiphidio cercariae were
associated with other types of cercariae (furcocercous,
gymnocephalous, monotome and echinostome).
However, such types of double infections were rarely
observed.
Seasonality and periodicity: In general, amphistome,
echinostome, furcocercous, gymnocephalous,
monostome and xiphidio cercariae were found in all
seasons, but their infection % were found to vary
greatly. However, the maximum infection of cercariae
in their respective snail host was found only in the late
rainy or pre-winter season (Table II). But monostome

50
Choubisa
Melanoides tuberculatus
Cercaria tali
C. buckleyi
C. chauhani
C. srivastavi
C. trioculata
C. martini
C. diglandulata
C. chengi
A spidogaster sp.
(metacecaria)
Fig. 16. Different cercarial and matacercarial species which emerged from Melania snail species.
and xiphidio cercariae showed two peak periods of
infection viz., pre-winter and summer seasons. The
emergence of these trematode larvae from their snail
hosts was observed to be at peak in mornings, nominal
during noon and lowest in evening hours. However,
during night, furco and monostome cercariae also
emerged out but their numbers were relatively very low.
Host-specificity and behaviours: In the present study,
most of the cercariae showed stern host–specificity:
amphistome cercariae were released from genera of
Planorbidae family, echinostome cercariae from genera
of Lymnaedae family and monostome cercariae from
genera of Thiaridae family. But furcocercous and
xiphidio cercariae showed less host-specificity, and
were found with various incidences in almost all the
genera of each family (Table I).
Except furcocercous and monostome cercariae, other
cercariae were generally encysted after 10-30 min, in a
beaker containing tap water. At the resting stage, all
types of cercariae moved-like a caterpillar on the
bottom of beaker but in the active (swimming) state,
they move like a spermatozoa and their heads remained
towards up side. Only furcocercous cercariae showed
jerking movement and these are relatively very active
swimmers having strong phototropic, negative
geotaxic and thigmotaxic behaviours; monostome
cercariae also showed such behaviours (Table III). The
strongest phototrophic behaviour (maximum

Pathogenic trematode cercariae infecting freshwater snails in Rajasthan
51
Indoplanorbis excustus
Cercaria manteri C. pandei C. dalibaghensis
C. chauhani
Gyraulus convexiusculus
C. chungathi
Fig. 17. Different cercarial species which emerged from genera of Planorbidae snail family.
their habitats were healthy and non-polluted, and had
abundant food. In the southern Rajasthan, most of the
lentic and lotic aquatic habitats are perennial and non-
polluted, containing aquatic vegetation which provided
a healthy environment or ecosystem for the breeding
and increase in the population of different snail species.
On the basis of present survey, it was found that the
larval digenean (cercariae) infection in surface
dwelling snails in the lentic habitats, especially in the
perennial ponds or reservoirs, was higher than those of
bottom dwellers in the same habitat. The bottom t al.,
emergence and aggregation of cercariae towards light
source) was found in those cercariae, who had eye
pigments on their body such as in the amphistome,
furcocercous and monostome cercariae. In general,
cercariae having large-sized body (except
furcocercous) were passive swimmers. Behaviours of
different cercariae have been hown in Table III.
DISCUSSION
In the present study, a rich diversity of snail species
(intermediate hosts) was found which indicated that

52
Choubisa
Lymnoea luteola
Lymnaea acuminata
Tetracotyle lymnaei
Cercaria indicae LXXIV Echinostome sp. Cercaria VII kerala
C. itoi
Cercaria talensis
Fig. 18. Different cercarial species which emerged
from L. luteola snail species.
Fig. 19. Different cercarial and matacercarial species
which emerged from L. acuminita snail species.
dwellers of the lentic habitats showed greater infection,
as compared to their counter-parts in the lotic habitats.
This has occurred probably because the chances of
contact between free-swimming miracidia and surfacedwelling
snail species were higher, and thus the lentic
species were found to be infected maximally, compared
to the bottom-dwelling species such as snails of
Planorbidae, Viviparidae and Thiaridae family as they
had little chances of contact with miracidia of various
species. Running water (lotic habitat) diminishes the
chance of interaction between miracidia and snails, and
consequently minimizes the incidence of larval
infection. Similar observations have been made for
cercarial infection in lotic waters (Radke et al., 1961;
Babikar et al., 1984; Choubisa, 1986a,b). These
workers concluded that the infectivity was inversely
proportional to the velocity of water flow but the
diversity of different cercariae is much more dependent
upon the member of different final hosts, which inhabit
and frequently visit the water bodies as well as the
density of miracidia and the population-size of snails
species.
The emergence of two types of cercariae from a single
snail host is referred to as a case of double infection.
Such cases were rarely observed in the present study.
Such type of infections have also been observed by
Mukherjee (1966), Jain (1970), Pandey and Agarwal
(1978) and Choubisa (1986b). It is possible because of
the identical host-specificity of two different digentic
trematode parasites.
Most of the cercariae emerge through out the year or in
the all reasons with varying incidence of infection.
However, the maximum incidence of infection was
found in the late rainy or pre-winter season. It is a fact
that rain-water is responsible for the aggregation of
different types of miracidium larvae in the aquatic
habitats. Secondly, the rain-water increases the snail
population. Because almost all the fresh water snail
species breed in the rainy season, most of the cercariae
are released from their snail hosts in the late rainy
season or pre-winter season. Simlar observations have
also been reported by Mukherjee (1966), Mohandas
(1974), and Pandey and Agarwal (1978). These workers
have reported two peaks of cercariael infection in a
year, as has been observed in the present study.
However, looking at the variability in the cercarial
infection from place to place and year to year, it is
proposed that the peaks of cercarial infection with a

Pathogenic trematode cercariae infecting freshwater snails in Rajasthan
53
Table I. Intermediate snail hosts and their trematode cercarial larvae
Snail host species Habit/ Habitat Trematode cercarial larvae
(A) Lymnaeidae:
Amphi Echino Furco Gymno Mono Xiphi
(i) Lymnaea acuminata f. patula Sd/Le - ++ - ++ - ++
(ii) L. acuminata f. chlamys Sd/Le - ++ - ++ - ++
(iii) L. acuminata f. typical Sd/Le - ++ - ++ - ++
(iv) L. luteola f. austrails Sd/Le - ++ - ++ - ++
(v) L.luteola f. typical Sd/Lo - ++ - ++ - ++
(vi) L. luteola f. impure Sd/Lo - ++ - ++ - ++
(vii) L. luteola f. rufescens Sd/Lo - ++ - ++ - ++
(B)
Planorbidae:
(i) Indoplanorbis exustus Bd/Lo , Le ++ + ++ - - +
(ii) Gyraulous convexiusculus Bd/Lo, Le ++ + ++ - - +
(C) Viviparidae:
(i) Vivipara bengalensis race gigantea Bd/Lo , Le - + ++ - - ++
(ii) V. bengalensis race mandiensis Bd/Lo, Le - + ++ - - ++
(D) Thiaridae:
(i) Melania Striatella tuberculata Bd/Lo, Le - - ++ - ++ +
(ii) Faunus ater Bd/Lo, Le - - ++ - ++ +
(iii) M. (Plotia) scabra Bd/ Lo - - ++ - ++ -
(iv) Thiara ( Tarebia) lineata Bd/ Lo - - ++ - ++ -
++, commonly infected; +, rarely infected; -, non-infected; Bd, bottom-dweller; Le, lentic (stagnant water); Lo, lotic
(running water); Sd, surface dweller.
Amphi, amphistome; Echino, echinostome; Furco, furcocercous; Gymno, gymnocephalous; Mono, monostome; xiphi,
xiphidiocercaria.
Table II. Seasonal occurrence of trematode larvae (cercariae)
Types of cercariae *Snail species Seasons
Rainy Winter Spring Summer
(Jul-Sept) (Oct-Jan) (Feb-Mar) (Apr-Jun)
Amphistome Genera of Planorbidae ++ +++ + +
Echinostome Genera of Lymnaeidae + +++ ++ +
Furcocercous Genera of Viviparidae ++ +++ ++ +
Gymnocephalous Genera of Lymnaeidae ++ +++ + +
Monostome Genera of Thiaridae + +++ ++ +
Xiphidio Genera of Lymnaeidae + +++ ++ +
+++, > 90% cercariae infection; ++, 30-60% cerarial infection
+, < 30% cercariae infection; -, infection is not found; * as in Table-I.

54
Choubisa
Table III. Relative behaviours of cercariae
Types of cercariae
Active
Negative- Photo- Thigmotaxism
Swimming geotaxism tropism
Amphistome + + +++ -
Echinostome + + ++ -
Furcocercous +++ +++ +++ +++
Gymnocephalous + + + -
Monostome +++ +++ +++ ++
Xiphidio+ ++ + -
+++, More; ++, Moderate; +, less; -, no response .
particular snail species are not stable but vary due to REFERENCES
change in the physico-chemical factors of a particular Babiker A, Fenwick A and Amin MA. 1984 . Localization of
niche (Choubisa , 1986a). It is interesting to note that Biomphalaria pfeifferi by Schistosoma mansoni miracidia in
except furcocercous and xiphidio cercariae, others stagnant and running water under field conditions.
showed stern host-specificity. Such observations have Hydrobiologia 110:235-240.
rarely been observed and discussed (Choubisa 1986a, Cheng TC.1973. General Parasitology Academic Press, New
1991). However, such findings are more useful for York and London.
forecasting the endemicity of a particular trematodiasis
Choubisa SL. 1986a. The biology of certain larval trematods
disease in a particular area.
infecting fresh water snails of Lakes of Udaipur. Ph. D Thesis,
M. L. S. University, Udaipur.
Strong phototactic behaviour among different cercariae
is much more related to the presence of eye pigments on Choubisa SL. 1986b. Incidence of larval trematodes infection
their body (furcocercous, monostome and amphistome and their seasonal variation in the fresh water of molluscs of
cercariae). Active swimming of cercariae (furcocercous southern Rajasthan. Rec Zool Sur India 83:69-80.
and monostome) is associated with the morphological Choubisa SL. 1991. Comparative study on cercarial behaviours
adaptive characters such as small-sized main body, and their host specificity. Ind J of Parasit 15:125-128.
prominent spinosed and muscularized bifurcated fail
Choubisa SL. 1992. Molluscs as bio-indicators for the trophic
(furcocercous), and ventral and dorsal fins on tail stages of lakes and lotic environments. Bull of Pur Appl Sci
(monostome). However, furcocercous cercariae are 11:35-40.
relatively more active swimmers as compared to
Choubisa SL and Sharma PN. 1983. Seasonal variation of
monostome, although both types of cercariae are active
cercarial infection in snails of Fateh Sagar lake of Udaipur.
swimmers. Additionally, such cercariae are strong Ind J Parasit 7:111-113.
negative-geotaxic as they can move against the gravity.
Erasmus DA. 1972. The Biology of Trematodes. Printed in Great
The findings of the present study can be useful in the Britain at the University Press, Belfast.
preparation of future health plans in tribal regions for Clarke AA.1979. Gastropods as indicators of trophic lake stages.
the preservation of the health of tribals and their The Nautilus 94:138-142.
domesticated animals, in understanding the biology of
Harman WN.1974. Snails (Mollusca: Gastropoda). In: CW Hart
trematode parasites, and to significantly add to the
Jr. and S. L. H. Fuller (Eds.), Pollution Ecology of Fresh
existing knowledge of parasitology.
water Invertebrates (Academic Press, New York).
ACKNOWLEGEMENTS
Jain SP. 1970. Double infection of larval trematodes in molluscs.
Agra Univ J Res Sci 21:47-48.
I thank the University Grants Commission, New Delhi
Mohandas A. 1974. Studies on the fresh water cercariae of Kerala
for the financial assistance (F.No.4S-09/2004-
I. Incidence of infection and seasonal variation. Folia Parasit
2005(MRP/CRO)/203008). The author is grateful to 21:311-317.
Prof. P. N. Sharma (Parasitologist) for his astute advice
and valuable comments.
Mukerjee RP.1966. Seasonal variations of cercarial infections in
snails. J Zool Soc Ind 18:39-45.

Pathogenic trematode cercariae infecting freshwater snails in Rajasthan
55
Mukerjee RP. 1980. Collection and study of larval trematods Schistosoma mansoni cercaria released under laboratory and
(Platyhelminthes). Proc Workshop Tech Parasit Zool Sur Ind field conditions. Expl Parasit 11:323-331.
23-26.
Ray HC and Mukherjee I. 1963. Fauna of Rajasthan, India. Part 3
Pandey KC and Agarwal N. 1978. Larval trematodes and Mollusca. Rec Zool Sur Ind 61:403-436.
seasonal variations in snails of Kathauta Tal, Lucknow. Ind J
Parasit 2:119-120.
Tonapi GT. 1980. Fresh Water Animals of India. Oxford and I. B.
H. Publishing Company, New Delhi, India.
Radke MG, Ritchie LS and Rowan WB. 1961. Effects of water
velocities on worm burdens of animals exposed to

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 56-59
Original paper
J P D
On Paralueheia guptai n. gen. & n. sp. (Acanthocephala:
Plagiorhynchinae Meyer, 1931) from Stromateus sinensis
A. M. Saxena and Rahul Gupta
Department of Zoology, University of Lucknow, Lucknow.
Received 24 March 2008; revised 19 May 2008; accepted 10 September 2008
ABSTRACT. Paralueheia guptai n. gen. & n. sp. from the intestine of a marine fish Stromateus
sinensis Day, from Deegha, West Bengal, India, is described. As compared with other genera of
subfamily Plagiorhynchinae Meyer, 1931, the new genus was found to possess unique morphoanatomical
characters: proboscis having 20 longitudinal rows of hooks with 17–18 hooks per row;
testes pre-equatorial and tandem; two cement glands. Diagnostic characters of the new genus and a
key of genera are provided.
Keywords: Acanthocephala, Deegha, intestine, Paralueheia guptai, Stromateus sinensis Day.
INTRODUCTION
The Acanthocephala are endoparasites which lack a
digestive tract in all stages of their development. The
characteristic feature of the phylum is an organ of
attachment consisting of an invaginable proboscis,
armed with rows of re-curved hooks (Hyman, 1951).
Out of 800 known species, 229 (28.62%) of them have
been reported from India (Alfred, 1998), including 203
species native to Indian region (Bhattacharya, 1998).
This indicates a very high percentage of prevalence.
Fishes have been found to be more susceptible to
infection of Acanthocephala (Milton and Franson,
1999; Taraschewski, 2000). In the present paper, a new
genera and species is described as Paralueheia guptai
n. gen. & n. sp. (Fig. 1–4) from the intestine of a marine
fish Stromateus sinensis Day (Fig. 5), recovered at
Deegha, West Bengal, India.
MATERIALS AND METHODS
A large number of marine fishes were collected from the
Corresponding author: Dr. A. M. Saxena, Department of
Zoology, University of Lucknow, Lucknow-226 007, India.
Email: anandmsaxena@rediffmail.com
sea coast of Deegha, West Bengal, and dissected out and
examined. During the examination, two male
Acanthocephala specimens were recovered from the
intestine of S. sinensis. The parasites were thoroughly
washed and then kept in refrigerator till complete
reversion of proboscis. Later, the parasites were
flattened under slight pressure of cover glass and kept in
A. F. A. fixative (50% alcohol, formaline and acetic acid
in ratio of 100:6:2.5) for 24 h. After fixation, the
parasites were stained in acetoalum carmine,
differentiated in acid alcohol, dehydrated through
ascending series of alcohol, cleared in xylol and then
mounted in Canada balsam. Diagrams were made with
the aid of Camera Lucida. All measurements are in
millimeter.
Type Description: Body is elongate, spinose,
cylindrical and slightly broader at middle and narrow at
extremities. Size is 4.2–10.6 mm long and 0.60–1 mm
wide. Proboscis is sub-globular, short, 0.56–1.2 mm
long and 0.38–0.66 mm wide, and having 20
longitudinal rows of hooks with 1–18 hooks per row.
Proboscis hooks are not uniform. Hooks of anterior
region are 0.060–0.062 mm long, of middle region

A n. gen. & n. sp. from Stromateus sinensis
57
Fig. 1-4. Paralueheia guptai n. gen. & n. sp.
Fig. 1. Entire male (ventral view).
Fig. 2. Male proboscis enlarged (ventral view).
Fig. 3. Single row of proboscis hooks of male.
Fig. 4. Proboscis hooks enlarged.
0.052–0.056 mm long and of posterior region number, long, tubular, and 0.96–2.17 mm long and
0.041–0.048 mm long. Neck is short and 0.18–1.06 mm 0.06–0.14 mm wide. Seminal vesicle is pyriform, and
long. Trunk is spinose at anterior region, cylindrical, 0.22–0.33 mm long and 0.07–0.25 mm wide. Bursa is
and 3.46–7.9 mm long and 0.60–1 mm wide. Proboscis globular or pyriform, without bursal rays, and
receptacle is double walled and claviform, and 0.31–0.52 mm long and 0.16–0.34 mm wide. Genital
1.05–3.15 mm long and 0.22–0.26 mm wide. Lemnisci pore is terminal.
are equal, extending up to middle of anterior testis, and
0.56–1.1 mm long. Testes are ovoid or elongate,
DISCUSSION
tandem, pre-equatorial, subequal, and apart from each The present form of the parasite has been referred to
other. Anterior testis is 0.14–0.21 mm long and subfamily Plagiorhynchinae Meyer, 1931 of the family
0.08–0.16 mm wide, and at 1.96–2.89 mm from Plagiorhynchidae Golvan, 1960 in having numerous
anterior end of the trunk. Posterior testis is 0.2–0.21 mm hooks on sub-globular proboscis; lemnisci slender,
long and 0.09–0.17 mm wide, and at 2.11–3.10 mm large and without distinct central canal, and cement
from anterior end of the trunk. Cement glands are two in

58 Saxena and Gupta
Fig. 5. Stromateus sinensis Day
Table I. Comparative chart of the genera of the subfamily Plagiorhynchinae Meyer, 1931
Genus Plagiorhynch Lueheia Oligoterorhync Pseudogordiorhync Paralueheia new
Structures us Luhe, 1911 Travassos, hus Monticelli, hus Golvan, 1957 genus
1919 1914
Body shape Plump, Fusiform Elongate Elongate
Elongate
fusiform
Proboscis Cylindrical or Subglobular, Ovoid, 14 Tunicated, 28-34 Subglobular,
and subcylindrical, 22-30 longitudinal longitudinal rows 20 longitudinal
proboscis 18-22 longitudinal rows with with 12-13 hooks rows with 17 to
hooks longitudinal rows with 6-7 hooks per row 18 hooks per row
rows with 8-10 hooks per row
10-12 hooks per row
per row
Proboscis Subcylindrical Claviform Subcylindrical Elongate Claviform
receptacle
Lemnisci Unequal, Unequal, Equal, Equal, Equal,
branched, unbranched, unbranched, unbranched, unbranched,
slender, 2 tubular, 4-6 tubular, 2 flattened, 2 tubular,2
Testes Preequatorial Preequatorial Postequatorial, Preequatorial, Preequatorial,
continuous tandem continuous
tandem
Cement 4, 6 or 8 in 4 in number 4 in number 3 in number 2 in number
glands number
glands tubular. Subfamily Plagiorhynchinae comprises proboscis hooks with 17–18 hooks per row. There are
of the following genera: Plagiorhynchus Luhe, 1911; 18–22 longitudinal rows of proboscis hooks with 8–10
Pseudogordiorhynchus Golvan, 1957; Lueheia hooks per row in Plagiorhynchus, 22–30 longitudinal
Travassos, 1919 and Oligoterorhynchus Monticelli, rows with 8–10 hooks per row in Lueheia, 14
1914 (Yamaguti, 1961). The present form differs from longitudinal rows with 6–7 hooks per row in
all these genera of subfamily Plagiorhynchinae in Oligoterorhynchus, and 28–34 longitudinal rows with
having two cement glands, and 20 longitudinal rows of 12–13 hooks per row in Pseudogordiorhynchus. There

A n. gen. & n. sp. from Stromateus sinensis
59
are 4–8 cement glands in Plagiorhynchus, three in with 8–10 hooks per row…… Lueheia Travassos,
Pseudogordiorhynchus, four each in Lueheia and 1919
Oligoterorhynchus.
Proboscis having 28–34 longitudinal rows of hooks
The present form can be further distinguished from with 12–13 hooks per row …
O l i g o t e ro rh y n c h u s , P l a g i o rh y n c h u s a n d Pseudogordiorhynchus Golvan, 1957
Pseudogordiorhynchus in having sub-globular
proboscis; from Lueheia in having lemnisci two and
ACKNOWLEDGMENT
equal, and testes tandem; from Oligoterorhynchus in Authors are thankful to Professor Nirupama Agarwal,
h a v i n g t e s t e s p r e q u a t o r i a l , a n d f r o m Head, Department of Zoology, University of Lucknow,
Pseudogordiorhynchus in having testes tandem (Table Lucknow for her constant encouragement and guidance
I). Accordingly, it is not possible to assign the present in this work.
form under any genus of the subfamily
Plagiorhynchinae. Therefore, it deserves the status of a REFERENCES
new genus Paralueheia, with type species P. guptai n. Alfred JRB. 1998. Faunal Diversity in India-An Overview. In:
gen. & n. sp., proposed for its reception.
Faunal Diversity in India. Volume I-VIII, ENVIS Centre,
Zoological Survey of India, Calcutta, India, pp. 1-495.
The new species is named in the honour of late
Bhattacharya SB. 1998. Faunal diversity in India. In:
professor S. P. Gupta, D. Sc., Department of Zoology,
Acanthocephala. Envis Centre, Zoological Survey of India,
University of Lucknow, Lucknow. Calcutta. India, pp. 93-98.
Paralueheia n. gen.
Golvan YJ. 1957. Acanthocephales d'oiseaux. Sixieme note:
Deux especes nouvelles parasites d'oiseaux d'Afrique
Generic diagnosis: Family-Plagiorhynchidae; occidentale Française: Gordiorhynchus (Gordiorhynchus)
subfamily-Plagiorhynchinae; body elongate, spinose gendrei n. sp. et Pseudogordiorhynchus antonmeyeri n. g. n.
and cylindrical. Proboscis subglobular, having 20 sp. Annals de Parasitology Hum Comp 32:83-97.
longitudinal rows of hooks with 17–18 hooks per row, Golvan YJ. 1960. Le Phylum des Acanthocephala (3. note). La
hooks large at apex and small at the base of proboscis. classe de Palaeacanthocephala Meyer, 1931. Annals de
Proboscis receptacle double-walled and claviform. Parasitology Hum Comp 35:138-165.
Lemnisci two, equal, tubular, and extending up to
Hyman LH. 1951. Acanthocephala, Aschelminthes and
middle of anterior testis. Testes ovoid or elongate, Entoprocta. In: The Invertebrates, Volume-3, First ed.,
subequal, preequatorial, and tandem. Cement glands McGraw-Hill Publishers, USA. pp. 1-49.
two, long and tubular. Seminal vesicle pyriform. Bursa
Luhe M. 1911. Acanthocephalen, Brauer Susswasserfauna
globular or pyriform without bursal rays. Genital pore
Deutschlands. Heft, pp. 16.
terminal. Parasite in marine fishes.
Meyer A. 1931. Die Acanthocephalen der arktischen Gebietes.
Type species: Paralueheia guptai n. gen. & n. sp. Fauna Arctica 6:9-20.
Key to the genera of the subfamily Plagiorhynchinae
Meyer, 1931
Milton F and Franson JC. 1999. Acanthocephaliasis. In: Field
Mannual of Wildlife Disease, US Department of Interior, US
Geology Survey, US, pp. 241-243.
1. Cement glands 2…… Paralueheia n. gen. & n. sp. Taraschewski H. 2000. Host-parasite Interactions in
Cement glands more than 2… 2
Acanthocephala: A Morphological Approach. Advances in
2. Testes preequatorial …… 3
Parasitology 46:1-179. \
Travassos, L. 1919. Informacoes sobre o material
Testes postequatorial …… Oligoterorhynchus helminthologico colleccionado na Ilha da Trinidade em,
Monticelli, 1914 1916. Arquivos do Museu Nacional Rio de Janeiro 22:159-
(Yamaguti, 1963)
167.
Yamaguti S. 1963. Oligoterorhynchus Monticelli, 1914. In:
3. Lemnisci branched …… Plagiorhynchus Systema Helminthum-Acanthocephala. Volume-5, John
Luhe, 1911 Wiley & Sons, New York, pp 91.
Lemnisci unbranched …… 4
4. Proboscis having 22–30 longitudinal rows of hooks

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 60-63
Original paper
J P D
Some epidemiological aspects of intestinal cestodes of
sheep in a temperate climate
K. A. Tariq, M. Z. Chishti and F. Ahmad
Post Graduate Department of Zoology, The University of Kashmir, Srinagar.
Received 5 June 2008; accepted 10 September 2008
ABSTRACT. The seasonal prevalence of intestinal cestode parasites in association with sex, breed
and age of sheep, from February 2005 to January 2007 in Kashmir valley was studied.
Gastrointestinal tract examinations of slaughtered sheep were conducted monthly to monitor the
seasonal prevalence of cestodes. During the period of two years, a total of 498 sheep were examined
and 178 of them were found positive for infection. The overall prevalence of the parasites of
st
nd
anoplocephalidae family was 35.7% (1 year: 34.05% and 2 year: 37.2%). The parasites in
decreasing order of prevalence were Moniezia expansa (6%), Avitellina centripunctata (3.7%), Stilesia
globipunctata (2.8%) and Thysaneizia giardi (2.2%). Highest infection was recorded in autumn
among the males of nomad breed (p > 0.05) vis-à-vis those of adult females of local breed. In
conclusion, we recorded quite low prevalence of anoplocephalidae in sheep of Kashmir valley
although season, age, sex and breed were the factors that influenced cestode infection in sheep.
Keywords: age, anoplocephalidae, breed, epidemiology, prevalence, sex, sheep
INTRODUCTION
Production of sheep is an attractive profession of
farmers in Kashmir valley, India, due to low capital
input and the ability of sheep to thrive on native
meadows and agricultural fields. There being limited
scope for industrialization in the valley, varied
activities of agriculture including sheep-rearing has
become a major source of income. Almost every rural
household rears sheep, which caters to their daily needs
of milk, meat, wool, leather and valuable organic
manure. Sheep are subjected to infection of diverse
types of helminth parasites particularly the soil- and
grass-transmitted species because of their grazing
habits. Anoplocephalids (intestinal cestodes) form an
Corresponding author: Mr. K. A. Tariq, Parasitology Research
Laboratory, Post Graduate Department of Zoology, The
University of Kashmir, Srinagar-190 006, India.
E-mail: drkatariq@yahoo.co.in
important group of intestinal helminth fauna of sheep.
The life cycles of intestinal cestodes in sheep are
indirect and herbage mites (Galumna, Oribatula,
Peloribates, Scheloribates, etc.) of the family
Oribatidae and psocids act as their intermediate hosts.
During feeding, the mites and psocids accidentally
ingest eggs of the tapeworms present in the faecal
matter and on the grass, and the larval stage called a
cysticercoid develops in them. Sheep become infected
by ingesting herbage containing mites carrying the
infective stage of the parasites (Soulsby, 1982).
Planning of control strategies of helminthosis requires
knowledge of the epidemiological factors that
influence the risk of helminth infections. To-date no
detailed epidemiological work has been carried out on
intestinal cestodes of sheep in Kashmir valley, hence we
took up the present study. The objective of this study
was to investigate the epidemiological aspects of

Intestinal cestodes of sheep
61
intestinal cestode parasites of sheep reared under
traditional husbandry conditions in Kashmir valley.
MATERIALS AND METHODS
This study was carried out in sheep slaughtered in
various abattoirs of the Kashmir valley from February
st
st
1 2005 to January 31 2007. A total of 498
gastrointestinal tracts of sheep were examined during
this period. All the sheep examined belonged to
Corriedale, Bhakarwal, Kashmir Merino and Kashmiri
breed. All the animals belonged to both sexes and their
age ranged from < one year to > four years. Monthly
collection of the samples was carried out to ensure the
seasonality of infection. The gastrointestinal tracts
carefully labeled with animal identification, breed, sex,
dental age and month of collection, were collected fresh
in normal saline (0.9%), and analyzed in the laboratory.
Abomasum, small and large intestines were analyzed
separately using standard procedures and the recovered
parasites were identified on the basis of morphological
descriptions of Soulsby (1982).
RESULTS
The overall prevalence of anoplocephalidae in sheep
was 35.7% (year 1: 34.05% and year 2: 37.2%). The
parasites in decreasing order of prevalence were
Moniezia expansa (6.0%), Avitellina centripunctata
(3.7), Stilesia globipunctata (2.8) and Thysaneizia
giardi (2.2%). The data pooled for seasonal estimation
of cestode infection revealed maximum cestode
infection (Year 1: 41.6%; Year 2: 46.8%) in autumn and
lowest (Year 1:19.2%; Year 2: 22.4%) in winter (p >
0.05 (year 1: p = 0.254; year 2: p = 0.204). The seasonal
prevalence of infection has been shown in Table I.
Influence of sex and breed and age on the prevalence
of infection
The present study revealed that sex, breed and age of the
host influence the prevalence of intestinal cestode
infection in sheep (Table II). Comparatively, males
were more infected than females. The breed wise
investigation of infection revealed low prevalence in
Table I. Seasonal prevalence of intestinal cestode
parasites in sheep over a period of two years
Season Year Examined Infected (%)
Spring Year 1 60 19 (31.6)
Year 2 72 22 (30.5)
Summer Year 1 60 24 (40.0)
Year 2 72 29 (40.2)
Autumn Year 1 60 25 (41.6)
Year 2 64 30 (46.8)
Winter Year 1 52 10 (19.2)
Year 2 58 13 (22.4)
Mean ± SE Year 1 58±2.0 19.5±3.42; p = 0.254
Year 2 66.5±3.4 23.5±3.92; p = 0.204
local Kashmiri breed. The Bhakarwal and Corriedale
breed were more infected. The cross bred Kashmir
Merino was found to harbour intermediate level of
infection. However, the observations were statistically
insignificant (p > 0.05). The age wise observations
revealed highest prevalence in lower age groups.
Generally, less than one year old animals were more
infected and with the increase in age, the infection level
decreased.
DISCUSSION
Percentages with their respective means ± SE (standard
error of the mean) were employed to calculate the
prevalence of anoplocephalidae infection. Chi-square
test was employed to measure association between the
parasitism and season, age, sex and breed, and the
values were considered significant at p < 0.05. The data
was analyzed using statistical packages MINITAB-
13.20 for windows.
The observations of low prevalence of
anoplocephalidae seem to be in relation to the agro
climatic conditions of the valley. Similar parasites with
different prevalence's have also been reported in other
parts of the country and world under similar or different
climates. Kiran et al. (2005) found M. expansa as the
most prevalent helminth in sheep in Dehradun. Munib
et al. (2004) reported the overall prevalence of 28% of
M. expansa, M. benedeni and A. centripunctata.
Muraleedharan (2005) reported 1.65% prevalence of
Moneizia spp. in sheep. In Turkey, Aydenizoz and
Yildiz (2003) also reported low prevalence of M.
expansa (3.98%) and A. centripunctata (0.86%) in
sheep. M. expansa, M. benedeni, T. giardi and A.
centripunctata were also recovered from sheep in
Moldova (Zgardan, 2002).
The present study revealed definite seasonality of
infection and in line with this seasonal trend are the
observations of various authors (Soulsby, 1982;
Courteny et al., 1985). In Turkey, highest infection of
M. expansa, A. centripunctata, Thysaneizia ovilla in
sheep was observed in July (9.89%) and the lowest in
September (1.32%; Aydenizoz and Yildiz, 2003). The
over-wintering of mites on pastures and subsequent

62 Tariq et al.
Table II. Influence of sex, breed and age on infection over a period of two years
Factor Level Year 1 Year 2
Examined Infected (%) p-value Examined Infected (%) p-value
Sex Male 112 39(34.8) 0.792 140 50(35.7) 0.926
Female 120 39(32.5) 126 44(34.9)
Mean ± SE 116±4.0 39±0.0 133±7.0 47±3.0
Breed Corriedale 64 23(35.9) 69 27(39.1)
Kashmiri 50 16(32.0) 59 17(28.8)
Bhakarwal 67 24(35.8) 0.944 76 31(40.7) 0.682
Kashmir Merino 51 15(29.4) 62 19(30.6)
Mean ± SE 58±4.37 19.5±2.32 66.5±3.79 23.5±3.30
Age 4 35 10(28.5) 41 11(26.8 )
Mean ± SE 46.4±3.77 15.6±2.15 53.2±5.42 18.8±3.91
infection of animals in the following grazing months et al., 2004; Wildeus and Zajac, 2005) where some
may account for high prevalence in autumn season. As breeds were found to be more susceptible to helminth
the whole Kashmir valley remains snow covered in infection as compared to the others.
winter season, therefore, no fresh infection with these
anoplocephalidae occurs in winter. The infection to
The higher infection in lower age groups may be due to
sheep might be occurring somewhere in spring season
the low resistance in young animals. It is a well
and early summer every year when the animals are sent
established fact that lambs are more prone to cestode
for grazing in the pastures and agricultural lands. After
infection particularly in case of M. expansa and become
completing the required prepatent period in sheep, the
infected very early in life (Soulsby, 1982). A high
infection starts appearing in highest numbers in the
incidence of enterotoxaemia has been associated with
autumn season of respective years as was significantly
Moneizia infections in lambs in USSR (Vibe, 1976).
observed during the two years of the present study.
The low level of parasitism reported in adult animals is
due to the development of significant immunity.
The influence of sex on the susceptibility of animals to Similar findings are available from other studies also.
infections could be attributed to genetic predisposition. For example, in different age groups of sheep, young
Our results are in agreement with Gorski et al. (2004) animals were more prone to infection with intestinal
who reported that males were reported to be more cestodes than adult animals (Rauf et al., 2005).
infected. However, many studies have indicated that
females were more infected than males (Patel et al.,
In conclusion, the present endeavour reported quite low
2001; Lateef et al., 2005). Therefore, sex of the animals
prevalence of anoplocephalidae in sheep under
cannot be regarded as a significant factor for cestode
temperate agro climatic conditions of Kashmir valley
infection; however, local management conditions can
and we found season, age, sex and breed as important
play a significant role in the occurrence of cestode
factors that influenced the risk of cestode infection in
infection in sheep despite the vice versa gender
sheep.
differences reported in several studies.
REFERENCES
The difference in the level of infection between
different breeds of sheep observed in the present study
are in agreement with the observations of other workers
in various parts of the world (Li et al., 2001; Amarante
Amarante AET, Bricarello PA, Rocha and Gennari SM. 2004.
Resistance of Santa Ines, Suffolk, and IIe de France sheep to
naturally acquired gastrointestinal nematode infections.
Veterinary Parasitology 120:91-106.

Intestinal cestodes of sheep
63
Aydenizoz M and Yildiz K. 2003. Prevalence of Muraleedharan K. 2005. Prevalence of gastrointestinal parasites
Anoplocephalidae species in sheep and cattle slaughtered in of live stock in dry zone of Karnataka. Journal of Veterinary
Kirrikale, Turkey, Revue Med. Vet. 154:767-771. Parasitology 19:31-33.
Courteny CH, Parker CF, McClure KE and Herd RP. 1985. Patel MD, Nauriyal DS, Hasnani JJ and Gupta RS. 2001.
Resistance of exotic and domestic lambs to experimental Prevalence of gastrointestinal parasitism in goats maintained
infection with Haemonchus contortus. International Journal under semi-intensive and field management systems. Indian
of Parasitology 15:101-109. Journal of Veterinary Medicine 21:99-101.
Gorski P, Niznikowski R, Strzelec E, Popielarczyk D, Gajewska Rauf U, Lateef M and Sultana A. 2005. Prevalence of different
A and Wedrychowicz H. 2004. Prevalence of protozoan and species of tapeworm in sheep. Journal of Plant and Animal
helminth internal parasite infections in goat and sheep flocks Science 15:53-55.
in Poland. Arch Tierz Dummerstorf 47:43-49.
Soulsby EJL. 1982. Helminths, Arthropods and Protozoa of
Kiran S and Kumar SK. 2005. Prevalence of gastrointestinal
th
Domesticated Animals, 7 ed. The English Language Book
helminths in sheep and goats in Dehradun. Journal of Society and Bailliere Tindall, London, pp. 763-777.
Experimental Zoology 8:281-283.
Vibe VV. 1976. Parasitic coenurosis in sheep. Veterinariya
Lateef M, Iqbal Z, Jabbar A, Khan, MN and Akhtar MS. 2005. Moscow 6: 58-60.
Epidemiology of trichostrongylid nematode infections in
sheep under traditional husbandry system in Pakistan. Wildeus S and Zajac AM. 2005. Gastrointestinal parasitism in
International Journal of Agriculture and Biology 7:596-600. hair sheep and meat goat breeds grazing naturally infected
pasture, Sheep and Goat Research Journal 20: 42-46.
Li Y, Miller JE and Franke DE. 2001. Epidemiological
observations and heterosis analysis of gastrointestinal Zgardan E. 2002. Some aspects of the epidemiology and
nematode parasitism in Suffolk, Gulf coast native and Cross prophylaxis of Anoplocephalidosis in sheep in Moldovo.
bred lambs. Veterinary Parasitology 98:273-283.
Revista Romana de Parazitologie 12:82-83.
Munib H, Inamullah K, Muhsin S, Muhammad F and Ahmad N.
2004. Prevalence of Cestodes and Comparative Efficacy of
Various Anthelmintics in Rambouilliet Sheep. International
Journal of Agriculture and Biology 6:1128-1131.
Yadav AK and Tandon V. 1989. Gastrointestinal nematode
infections of goats in a subtropical and humid zone of India.
Veterinary Parasitology 33:135-142.

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 64-67
Short communication
J P D
Effect of the control of one-host cattle tick Boophilus
microplus on the growth and haematological parameters
of calves
S. Vatsya, R. R. Kumar, C. L. Yadav and R. Garg
Department of Veterinary Parasitology, G. B. Pant University of Agriculture and Technology, Pantnagar.
Received 8 June 2007; revised 8 August 2007; re-revised 1 October 2007; accepted 3 January 2008
ABSTRACT. A study was undertaken to understand the effect of the control of Boophilus microplus
ticks on live-weight gain and various haematological parameters in cattle calves. Following
application of Tickout (cypermethrin) @ 1 ml/liter, as spray wash, 94.68% control was attained, 3
st nd rd th th th th
days post-treatment. At the end of 1 , 2 , 3 , 4 , 5 , 6 and 8 week post-treatment (WPT), calves
showed a percent weight gain of 7.77, 13.54, 25.42, 41.29, 62.42, 80.10 and 100.54, respectively.
st nd rd th th th th
However, at the end of 1 , 2 , 3 , 4 , 5 ,6 and 8 WPT, a percent weight gain of 3.97, 12.60, 23.68,
35.12, 49.84, 67.76 and 87.80, respectively, was recorded in the untreated control calves. Tick
infestation caused anaemia in calves. Acaricide treatment resulted in the elevation of haematological
parameters, which reflected an effort on the part of calves to return to normal as compared to those in
control group in which persistence of anaemia was evident.
Keywords: Boophilus microplus, control, haematological parameters, weight gain
Ticks, especially Boophilus microplus, are
economically important pests of cattle in a tropical
country like India. Despite the fact that animals remain
infested with ticks throughout the year and have heavy
tick burden during peak tick-season resulting in great
production loss, it is treated as a casual entity. Though
very few studies have been carried out regarding the
impact of tick infestation on live-weight gain and
effects on haematological parameters in cattle in
different parts of the world (Johnnston et al., 1981;
Williams and Buckner, 1977), such reports were
difficult to be fished out from India. For this reason
only, the present study was undertaken and the results
are reported herein.
Corresponding author: Dr. C. L. Yadav, Department of
Veterinary Parasitology, G. B. Pant University of Agriculture
and Technology, Pantnagar-263 145, India.
Twenty two crossbred male calves (Jersey x Sahiwal;
28-31 day old, weighing 22–32 kg), naturally-infested
with B. microplus and maintained at Instructional Dairy
Farm, Pantnagar, were used in this study. The animals
were kept in yards made of concrete floors, and were
given standard feed. The calves were randomly divided
into two groups of eleven animals each. The animals of
group-I (G-I) were treated with Tickout (cypermethrin;
Alembic Veterinary Division, Vadodara) @ 1 ml/litre as
spray wash and their shed @ 2ml/litre. The animals of
group-II (G-II) were left untreated but kept separately
in a different shed. Side counts of ticks were made
(Holdsworth, 2006) before treatment, and on 3, 7 and
thereafter at 15 days interval, post treatment. The
animals were weighed and their blood was collected for
haemoglobin (Hb), packed cell volume (PCV), total
erythrocyte count (TEC), MCV, MCH and MCHC as
st
described by Jain (1993) at the start of the trial and 1 ,

Effect of the control of Boophilus microplus
65
Table I. Haematological alterations (Mean± SE) following treatment of cattle naturally infested with B. microplus
Weeks post treatment
Parameter 0 I II III IV V VI VII
Hb Treated 94.5±3.1 97.2±2.4 100±2.3 102.7±1.7 108.2±2.6 108.1±2.2 114.5±2.8 115±2.0
(g/l) Control 93.6±2.8 91.8±1.8 89.5±1.9 85.0±1.3 82.7±1.6 78.1±1.0 77.7±1.2 74.1±1.5
PCV Treated 29.27±0.97 30.45±0.95 31.0±0.71 31.45±0.66 33.0±.082 33.64±0.72 34.45±1.03 37.27±0.88
(%) Control 28.9±0.78 28.36±0.82 27.63±0.83 26.91±0.49 27.1±0.59 23.91±0.51 24.1±0.56 23.0±0.59
TEC Treated 5.89±0.19 5.94±0.15 5.90±0.11 5.94±0.14 6.31±0.17 6.43±0.14 6.48±0.26 6.62±0.19
12
(10 /1) Control 5.56±0.14 5.48±0.19 5.39±0.12 5.30±0.09 5.27±0.12 5.10±0.02 5.06±0.03 5.05±0.02
MCV Treated 49.89±1.52 51.15±0.16 52.64±1.10 53.01±1.01 52.37±0.78 52.54±1.65 53.64±1.17 56.52±0.98
(fl) Control 52.01±0.83 51.66±1.03 50.77±0.87 50.97±1.19 51.49±0.77 47.42±0.92 47.55±0.95 45.38±0.99
MCH Treated 16.01±0.47 16.36±0.13 16.950.19 17.31±0.25 17.160.22 16.89±0.5 17.25±0.43 17.46±0.33
(pg) Control 16.83±0.26 16.73±0.22 16.62±0.12 16.05±0.16 15.69±0.12 15.51±0.14 15.34±0.19 14.67±0.23
MCHC Treated 313.7±11.2 320.2±3.7 323.0±5.8 327.3±5.8 328.0±4.0 321.7±2.6 321.3±5.0 309.0±2.8
(g/l) Control 326.2±2.8 324.7±4.8 325.0±5.4 316.8±6.9 305.7±2.9 327.7±3.6 323.6±5.4 322.9±4.3

66
Vatsya et al.
nd rd th th th th
2 , 3 , 4 , 5 , 6 and 8 week after treatment (WPT). Tick infestation affects appetite, digestion and
metabolism of animals, possibly by the secretion a
Following application of cypermethrin, 94.68% control
hepatotoxic compound (Jonsson, 2006). Lower weight
was attained 3 days post-treatment (DPT). The animals
gain has also been reported by Okello-Onen et al.
became free of tick infestation 7 DPT, and their coat
(2003) in un-dipped tick infested cattle. Sutherst et al.
became shiny. In the untreated group, the number of
(1983) observed that cattle heavily infested with ticks
st
ticks increased from 38.37±2.63 (1 week) to 102±3.15
gained weight rapidly after removal of tick burden, as
th
at the end of trial (8 week). These animals appeared compared to the un-infested animals. These researchers
weak, dull with rough coats.
calculated the mean value for the effect of damage in
th th
At the start of 7 week, two animals and by the end of 8 Bos taurus cattle as 1.37±0.25g/engorging tick, and that
week, 5 animals were found to be infested with 1±0.002 f o r B . Ta u r u s x B . i n d i c u s c a t t l e a s
nymphal stages of ticks in G-I. Therefore, all the 1.18±0.21g/engorging tick. Scholtz et al. (1991)
infested animals in both the groups were treated with estimated the effect of individual engorged tick
Tickout @ 1 ml/litre, at the end of experiment.
in Hereford, Bonsmara and Nguni breeds of cattle to be
In the treated group, the mean weight increased from 8.9, 8 and 8.6 g, respectively. Nguni breed carried least
th
27.54±0.84 kg to 55.23±0.72 kg by 8 WPT, with a
number of B. decoloratus (5.30) and suffered from least
percent weight gain of 7.77, 13.54, 25.42, 41.29, 62.42,
reduction in weight gain. Williams et al. (1977), while
80.10 and 100.54, respectively, at the end of
studying the effects of Gulf Coast tick on weights of dry
st nd rd th th th th
1 ,2 ,3 ,4 , 5 ,6 and 8 WPT (Fig. 1). A major
lot Hereford steers observed that, compared to control
difference in the weight of animals was observed at the
animals, high- and low-infested animals were 24 kg and
rd
14 Kg lighter, respectively. The growth rates of cattle
end of 3 WPT. In the G-II animals, the mean weight
were far better, besides reduction in the incidence of
increased from 27.45±0.77 kg to 51.55±0.65 kg. At the
st nd rd th th th th
tick-born diseases, when strategic tick control measures
end of 1 ,2 ,3 ,4 ,5 , 6 and 8 week, the percent
are adopted as compared to no tick control or control
weight gain were recorded to be 3.97, 12.60, 23.68,
coupled with pasture spelling (Johnston et al., 1981).
35.12, 49.84, 67.76 and 87.80, respectively.
35
Mean live weight gain (kg)
30
25
20
15
10
5
0
-5
1 2 3 4 5 6 7
Treated group
Control group
Weeks post treatment
Fig. 1. Live weight gain in cattle calves infested with Boophilus microplus following acaricide application.

Effect of the control of Boophilus microplus
67
The results of the haematological studies are presented
in Table-I. Following acaricide application, the
haematological values viz. Hb, PCV, TEC were found
elevated, which reflected an effort on the part of the
animal to return to normal. Persistence of anaemia in
the tick infested animal could be the result of blood-loss
due to blood sucking activity of ticks. Ram et al. (2004)
recorded mortality of 19 calves in a herd of 36 crossbred
B. microplus-infested calves in the age group of
6–18 months. The carcass of calves revealed severe
anaemia and emaciation. The tick-infested surviving
animals were extremely weak, anaemic, and anorectic
and failed to gain weight.
Association for the Advancement of Veterinary Parasitology
(WAAVP) guidelines for evaluation the efficacy of acaricides
against ticks (Ixodidae) on ruminants. Vet Parasitol 136:29-
43.
Jain NC. 1993. Essentials of Veterinary Haematology. Lea and
Febiger, Philadelphia, pp 417.
Johnston LAY, Haydock KP, Leatch G. 1981. The effects of two
systems of cattle tick (Boophilus microplus) control on tick
populations, transmission of Babesia spp. and Anaplasma
spp. on production of Brahman crossbred cattle in the dry
tropics. Aust J Exp Agric Anim Husbandry 21:256-267.
Jonsson NN. 2006. The productivity effects of cattle tick
(Boophilus microplus) infestation on cattle with
particular reference to Bos indicus cattle and their crosses. Vet
Parasitol 137:1-10.
The results of the present study suggest that ticks affect
weight gain as well as various haematological Okello-Onen J, Tukahira EM, Perry BD, Rowlands GJ, Nagada
parameters in cattle. These observations assume SN, Musisi G, Bode E, Heinonen R, Mwayi W and Opuda-
Asibo J. 2003. The impact of tick control on the productivity
significance when production performance of animals
of indigenous cattle under ranch conditions in Uganda. Trop
is affected due to reduced weight gains, and is reflected
Anim Hlth Prod 35:237-247.
while taking decisions in adopting control measures in
Ram H, Yadav CL, Banerjee PS and Kumar V. 2004. Tick
the event of a tick outbreak. Thus there is a need for
associated mortality in crossbred cattle calves. Indian Vet J
strategic control of tick infestation in animals so that the 81:1203-1205.
productivity of animals remains unaffected.
Scholtz MM, Spickett AM, Lombard PE, Eusulin CB. 1991. The
ACKNOWLEDGEMENTS
effect of tick infestation on the productivity of cows of three
breeds of cattle. Onderstepoort J Vet Res 58:71-74.
The authors are thankful to the Dean, College of
Sutherst RW, Maywald GF, Kerr JD, Stegeman DA. 1983. The
Veterinary and Animal Sciences, G. B. Pant University effect of cattle tick (Boophilus microplus) on the growth of
of Agriculture and Technology, Pantnagar, and Joint Bos indicus X B. taurus steers. Aust J Agric Res 34:317-327.
Director, Instructional Dairy Farm, Nagla, Pantnagar,
Williams RE, Hair JA and Buckner RG. 1977. Effects of Gulf
for their cooperation and help during the study period. Coast tick on blood composition and weights of dry lot
Hereford steers. J Eco Entomol 70:229-233.
REFERENCES
Holdsworth PA, Kemp D, Green P, Peter RJ, Bruin CDe, Jonsson
NN, Letonja T, Rehbein S and Vercruysse J. 2006. World

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 68-73
Short communication
J P D
Mode of nutrition in pathogenic trematode larvae (redia
and cercaria) which infect hepatopancreas of fresh water
snails (Mollusca: Gastropoda)
S. L. Choubisa
Department of Zoology, Government Post Graduate College, Sirohi.
Received 19 November 2007; accepted 2 September 2008
ABSTRACT. Histochemical procedures were employed to demonstrate various hydrolytic enzymes
in the pathogenic trematode larvae, rediae and cercariae in relation to the nutrition or digestion of the
food. Tegument of both rediae and cercariae were found to be endowed with acid phosphatase, β-
glucuronidase, esterase and lipase. These hydrolases were localized in the gut of rediae, but caecal gut
of cercariae of Echinostoma tewarii and Cercaria udaipuriensis contained only acid phosphatase with
low activity. Comparison between the nutrition of rediae and cercariae in the present study revealed
the former to be nutritionally advanced over the latter. Redia has been found to use both the routes to
obtain nutrition i.e., tegument and caecal gut, whereas cercariae use tegument as evidenced by
histochemistry of tegument. Localization of these hydrolases in other parts or organ systems of these
larvae has also been discussed.
Keywords: cercaria, hydrolases, nutrition, redia, trematode larvae
Although, a large number of histochemical tests have
been conducted in various larval forms of digenetic
trematodes by previous investigators (Fripp, 1966;
Cheng and Yee, 1968; Clark, 1968; Porter and Hall,
1970a,b; Choubisa, 1989), no systematic studies have
been conducted on the enzymes involved in the
digestion in these larvae. Sporocyst is devoid of any
kind of digestive tract and, therefore, nutrients are
absorbed through general body surface. Only rediae
and cercaria are endowed with a simple digestive tract.
Nevertheless, previous studies on nutrition of cercariae
have clearly revealed them to be non-feeding and their
caecae mostly devoid of digestive enzymes. Rediae of
most of the trematodes are provided with oesophageal
gland and long simple gut, and have received much
attention for the presence of various enzymes involved
Corresponding author: Dr. S. L. Choubisa, 205, Chanakyapuri,
Sector 4, Hiran Magri, Udaipur-313 002, India.
E-mail : choubisasl@yahoo.com
in nutrition (Cheng, 1964; Probert, 1966; Erasmus,
1972). The aim of the present study is to
histochemically localize the presence of certain
hydrolytic enzymes in some species of cercariae and
rediae, hitherto uninvestigated.
Two different unreleased cercariae, Echinostoma
tewarii. (Choubisa and Sharma, 1985) and
furcocercous cercariae, Cercaria udaipuriensis
(Sharma and Choubisa, 1983) were collected by
breaking the shell of fresh water snails Indoplanorbis
exustus and Vivipara bengalensis, respectively. The
former species is released from birth-pore of rediae,
whereas the letter directly from sporocysts. The size of
C. udaipuriensis is larger than that of E. tewarii, and
bears prominent oral sucker, pharynx, oesophagus,
broad and crenated intestinal caeca, and are active
swimmers. However, E. tewarii possesses these parts of
digestive system but caeca was narrow and
oesophagous surrounded by many penetration glands.

Mode of nutrition in pathogenic trematode larvae
69
Moreover, this species of cercariae has prominent
distribution of many cystogenous glands over the body.
The gut of rediae of E. tewarii contains brownish
yellow macrogranules and extends up to three-fourth of
the body.
Various histochemical methods for the localization of
certain enzymes such as lipase, acid phosphatase, -
glucuronidase and simple esterase in these trematode
larvae were used as earlier (Choubisa, 1988). The
control tissues were also run simultaneously to ensure
the correct localization of these enzymes.
The intensity of the activity of lipase, acid phosphatase,
β-glucuronidase and simple esterase in different tissues
of E. tewarii and C. udaipuriensis, as well as in rediae of
E. tewarii has been depicted in Table I and II. Only
intestinal ceaca of E. tewarii cercariae revealed the low
intensity of acid phosphatase and simple esterase
activity, whereas the caeca of C. udaipuriensis did not
show the activity of the above mentioned enzymes.
However, the gut of redia of E. tewarii was found to
have maximum activity of acid phosphatase and
esterase. The oesophageal gland of the present redia;
however, was found to be positive for glucuronidase,
lipase, esterase and acid phosphatase. The intense
activity of lipase was observed on the body wall,
cystogenous and penetration glands of echinostome
cerariae, E. tewarii (Plate 1; Fig. 3), whereas the
funcocercous cercariae also revealed the lipase activity
but moderate in the penetration and cystogenous
glands, body wall and excretory bladder (Plate 2; Fig. 4
and 5). The oral suckers of both cercariae also indicated
low activity of this enzyme. The activity of acid
phosphatase was seen in both types of glands,
penetration and cystogenous, as well as in excretory
bladder of both cercarial species. Low activity of this
enzyme was seen in intestinal ceaca only in the E.
tewarii (Plate 1; Fig. 2). Whereas β−glucuronidase
activity was not found in any part of the cercariae, on
the contrary, the esterase was seen in the from of bluish
micro-granules in various parts of cercarial body, its
maxium activity was observed in the nervous system
-4
(Fig. 1 in both Plate 1 and 2). The use of 10 M eserine
sulphate inhibited esterase activity completely in the
nervous system and to a great extent in cystogenous and
penetration glands, near ceaca. Except simple esterase
activity, no other enzymes viz., acid phosphatase, lipase
and β−glucuronidase revealed any activity in the tail of
both types of cercariae.
Table I. The activity of enzymes in redia and an unreleased cercariae, Echinostoma tewarii
Enzymes Cercaria (E. tewarii) Redia of E. tewarii
BW IC EB NS S T OG C
Acid phosphatase + + ++ - + + +++ +++
Lipase +++ - - - + - +++ -
β-glucuronidase - - - - - - +++ -
Simple esterase ++ + - +++ ++ ++ +++ +++
+++, intense activity; ++, moderate activity; +, low activity; -, no activity;
BW, body wall; C, caecum; EB, excretory bladder; IC, intestinal caeca; NS, nervous system; OG, oesophageal gland; S,
suckers; T, tail.
Table II. The activity of enzymes in an unreleased furcocercous cercariae, C. udaipuriensis
Enzymes
Cercaria (C. udaipuriensis)
BW IC EB NS S T
Acid phosphatase + + ++ - + -
Lipase ++ - - - + -
β-glucuronidase - - - - - -
Simple esterase ++ - - +++ ++ +++
+++, intense activity; ++, moderate activity; +, low activity; -, no activity;
BW, body wall; C, caecum; EB, excretory bladder; IC, intestinal caeca; NS, nervous system; OG, oesophageal gland;
S, suckers; T, tail.

70 Choubisa
OS
CC
Oe
PLNC
ExB
CR
ANC
PVNC
VS
OS
CyG
VS
ExB
Fig. 1. Localization of simple esterase in the cercaria, E. tewarii
revealing intense activity in the nervous system and moderate in
body wall, suckers and tail region (x 100).
Fig. 3. E. tewarii cercaria revealing the intense activity of lipase
in the body wall, cystogenous, pentration glands and in suckers.
(x 100).
Oe
M PhG
G
C
OS
G
M
CyG
Oe
Ph
VS
ExB
G
C
Fig. 2. Activity of acid phosphatase in the cercaria E. tewarii
showing moderate activity in the excretory bladder,
cystogenous and penetration glands and low in the body wall,
suckers and intestinal caeca. (x 100).
Fig. 4 and 5. Rediae of E. tewarii revealing the intense activity of
simple esterase (Fig. 4) in the oesophageal gland and gut. (x
100).

Mode of nutrition in pathogenic trematode larvae
71
OS
CC
PLNC
PVNC
CyG
OS
IC
T
ExB
FR
(Fig. 1) β-glucuranidase
(Fig. 3) and lipase
OS
OS
ExB
FR
IC
Fig.4
T
OS
IC
ExB
T
(Fig. 2), acid phosphatase
(Fig.4 and 5) activity in various regions.

72 Choubisa
From the present study, it is evident that rediae of E.
tewarii feed upon the hepatopancreatic tissue. The cell
debris of the pancreas has been observed in the caecum
of the rediae. Histology of the pathological tissue
clearly supports tissue feeding of rediae.
Certain hydrolytic enzymes such as acid phosphatase,
ATPase and simple esterase have revealed their
presence in the gut of the rediae in the present study. The
oesophageal gland of the redia; however, was found
positive for lipase, -glucuronidase, esterase and acid
phosphatase enzymes. Because no activity of lipase and
-glucuronidase was observed in the caecum, it appears
that these two enzymes are specially secreted by
oesophageal glands for extracellular digestion of the
food, the host tissues.
Results of the present investigation corroborate the
findings of Cheng (1964) and Probert (1966), who have
also reported the ceacal wall to be very rich in acid
phosphatase and non-specific esterase in their studies
on rediae. The presence of phosphatases in the ceacal
gut may not be directly involved in the digestion and
absorption of nutrients, in view of the strong criticism
by Reed (1966). Nevertheless, presence of cellular
debris and multitude of digestive enzymes conclusively
prove the redial gut to be an active site for the digestion
of host cells.
Although the redia in this study possesses a gut and a
battery of enzymes for the digestion of a variety of
nutrients such as proteins, glycogen and lipids present
in the host tissues and taken as the food material, it does
not preclude the possibility of obtaining the nutrients
through tegument, as do the sporocysts and adult flukes.
This contention gets support from the presence of
phosphatases on the tegument of redia, as well as, the
ultrastructural studies of the redia of Parochis acanthus
(Rees, 1966) and Neophasis lageniformis (Koie,
1971a), in which their external tegument revealed the
presence of microvilli and intense surface folding,
respectively. Thus the body wall of the present redia
may be regarded as analogous to the surface
amplification that occurs at the outer surface of tape
worms and sporocysts (Whitfield, 1979).
When the echinostome and furcocercous cercariae of
the present study were tested for the histochemical
distribution of various hydrolytic enzymes in tegument
and gut, it was found that except acid phosphatase no
other enzyme gave positive activity. The absence of an
array of enzymes in the gut, except acid phosphatase,
clearly indicated the absence of digestion of food in this
larval stage. The absence of most digestive enzymes in
the caecae of cercariae are conducive to their being a
non-feeding stage. Koie (1971b) has, however, reported
the histochemical presence of some hydrolytic
enzymes in the intestinal cells of the cercaria
Zoogonoides viviparous, whereas Stirwalt and Walters
(1964) have demonstrated aminopeptidase activity in
the intestinal caeca of Schistosoma mansoni cercariae.
The latter has, however, not discussed the significance
of the enzyme in relation to larval nutrition.
Acid phosphatase has been established as a marker for
lysosome (de Duve, 1963), and the lysosomes play an
important role in the intracellular as well as
intercellular digestion of several flat worms. Because
cercariae in the present study are non-feeding larval
stages and their caecal cells contain acid phosphatase,
it is possible that the caecal cells during emergency
(i.e., when glycogen reserve of the cercariae has
exhausted) undergo sequestration in order to provide
the threshold energy for the survival of the cercariae
till it has reached the host and started obtaining
nutrition from there. A similar suggestion has been
made by Sharma and Mandawat (1982) for high
concentration of acid phosphatase present in the
caecal cells of the starving Ganeo tigrina recovered
from hibernating frog host.
Tegument of cercariae appears to compensate the
function of the uptake of nutrients by non-functional
intestinal caecae. In view of this contention, occurrence
of certain hydrolytic enzymes such as acid phosphatase,
lipase, -glucuronidase and simple esterase in the
tegument of unreleased as well as free living cercariae
E. tewarii and C. udaipuriensis are significant from
permeability of nutrients point of view. These results
are similar to the findings of the other investigators who
have advocated the role of phosphatases in the
translocation of nutrients through the tegument of the
cercariae of Zoogonoides viviparus, S. mansoni and
Cloacitrema narrabeensis etc. (Stirewalt and Walters,
1964; Dixon, 1970; Koie, 1971a). Although cercariae
are advanced over rediae but from nutritional point of
view this appears to be vice versa.
The present study on the nutrition of redia and cercariae
delineated a sharp contrast. The comparison revealed
the redia to be nutritionally advanced over cercaria.
And this seems to be an excellent adaptation and high
strategy on the part of redia to obtain more nutrition so
that more protein and other nutrients are made available
to the developing cercariae within the body of the redia.

Mode of nutrition in pathogenic trematode larvae
73
ACKNOWLEDGEMENTS
The author is grateful to Prof. P. N. Sharma (Eminent
Parasitologist) for astute advice and comments.
Koie M. 1971a. On the histochemistry and ultrastructures of the
redia of Neophasis lageniformis (Lebour, 1910) (Trematoda,
Acanthocolpidae). Ophelia 9:113-143.
Koie M. 1971b. On the histochemistry and ultrastructure of the
REFERENCES
tegument and associated structures of the cercaira of
Zoogonoides viviparus in the first intermediate host. Ophelia
Cheng TC. 1964. Studies on phosphatase systems in 9:165-206.
hepatopancreatic cells of the molluscan host of
Echinoparyphium sp. and in the redia and cercaria of this
trematode. Parasitol 54:73-79.
Cheng TC and Yee HWF. 1968. Histochemical demonstration of
amino peptidase activity associated with the intra-molluscan
stages of Philophthalmus gralli Mathis and Leger. Parasitol
58:473-480.
Choubisa SL. 1988. Histological and histochemical observations
on the digestive gland of Melanoides tuberculatus
(Gastropoda) infected with certain larval trematodes and
focus on their mode of nutrition. Proc Indian Acad Sci (Anim
Sci) 97:251-262.
Choubisa SL. 1989. Distribution of non-specific esterase in
certain larval digeneans with a note on morphology of
nervous system. Ind J Exp Biol 27:32-57.
Choubisa SL and Sharma PN. 1985. Cercaria tewarii n. sp.
(Echinostomatid cercaria) from fresh water snail,
Indoplanorbis exustus (Deshayes). Bio-Sci Res Bull 1:50-53.
Clark TS. 1968. Histochemical studies of glandular contents of
two sp. of cercairae. Am J Med Tech 34:446-452.
de Duve C. 1963. The Lysosome concept. In: A.V.S. de Renk and
M. P. Camerson (Eds). CIBA Foundation Symposium on
lysosomes. London. J.A. Churchill.
Dixon KE. 1970. Absorption by developing cercariae of
Cloacitrema nerabeenesis (Philophthalmidae). J Parasitol
56:416-417.
Erasmus DA. 1972. The Biology of Trematodes. Printed in Great
Britain at the University Press, Belfast.
Fripp PJ. 1966. Histochemical localization of β-glucuromidase
in Schistosomes. Exp Parasitol 19:254-263.
Porter CW and Hall JE. 1970a. Histochemistry of Cotylocerous
cercaria I. Glandular complex in Plagioporus lepomis. Exp
Parasitol 27:368-377.
Porter CW and Hall JE. 1970b. Histochemistry of a Cotylocerous
cercaria II. Hydrolytic and oxidative enzymes in
Plagioporous lepomis. Exp Parasitol 27:378-387.
Probert AJ. 1966. Histochemical studies on the rediae and
cercariae of Echinoparyphium recurvatum Linstow. Nature
210:550-551.
Reed CP. 1966. Nutrition of intestinal helminths. In : Biology of
Parasites. EJL Soulsby (Ed.), Academic Press. pp. 101-126.
Rees FG. 1966. Light and electron microscope studies of the
redia of Parorchis acanthus. Nicoll 56:589-602.
Sharma PN and Mandawat S. 1982. A comparison of
morphology in Ganeo tigrinum from hibernating and nonhibernating
Rana cyanophyctis and R. tigrina. J Helminthol
56:5-10.
Sharma PN and Choubisa SL. 1983. Cercaria udaipuriensis n.sp.
from fresh water snails, Vivipara bengalensis from Fateh
Sagar lake. Ind J Parasit 7:209-212.
Stirewalt MA and Walters M. 1964. Histochemical assay of
glands of cercarial of Schistosoma mansoni. J Parasitol 50
Suppl. No. 94:44.
Whitfield PJ. 1979. The Biology of Parasitism: An introduction
to the study of associating organisms. Edward Arnold
(Publisher).

Journal of Parasitic Diseases: June 2008, Vol. 32, No. 1, 74-76
Short communication
J P D
Amoebic dysentery in dogs and dog owners
1 2 2 3
Saleh Umair , Azhar Maqbool , Zubair Shabbir and M. D. Ahmad
1
Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
2
University Diagnostic laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan.
3
Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Received 15 September 2008; revised 19 September 2008; accepted 27 September 2008
ABSTRACT. A total of 100 samples each of faeces and serum of dogs, and of their owners were
collected for the detection of Entamoeba histolytica. E. histolytica, in feces, was examined directly as
well as by flotation method, whereas in case of extra luminal invasion their antibody level was
determined by latex agglutination test. The prevalence of E. histolytica in faeces of dogs and humans
was estimated to be 14% and 10%, respectively. The prevalence of extra luminal amoebiasis, as
determined by latex agglutination test which detected the antibodies in serum of dogs and their
owners, was estimated to be 12% and 8%, respectively.
Keywords: amoebiasis, latex agglutination, prevalence
The best known zoonotic species of amoebae
parasitizing humans is Entamoeba histolytica, the
causative agent of amoebic dysentery or amoebiasis.
An estimated 400–500 million people in the world are
infected with E. histolytica, and only 5% to 10% of
them show symptoms of the disease (Garcia and
Bruchner 1997). The usual mode of E. histolytica
infection is the ingestion of cysts from contaminated
hands, food or water, and the incidence of the disease
increase considerably in thickly populated areas where
contact with infected individuals is more likely. E.
histolytica exhibits a typical fecal-oral life cycle
consisting of infectious cysts passing out in the faeces,
and trophozoites which replicate within the large
intestine. The infection is acquired through the
ingestion of cysts, and the risk factors are similar to
those of other diseases transmitted by the fecal-oral
route; contaminated food and water are probably the
primary sources of infection. The higher prevalence in
Corresponding author: Prof. Dr. Azhar Maqbool, Department
of Parasitilogy, University of Veterinary and Animal Sciences,
Lahore, 54000, Pakistan. E-mail: salehumair@hotmail.com
areas of lower socioeconomic status is likely due to
poor sanitation and a lack of indoor plumbing.
However, a higher prevalence of E. histolytica infection
is also observed in institutions such as mental hospitals,
orphanages and prisons, where crowding and problems
with fecal contamination are the contributing factors.
The symptoms of amoebiasis range from mild
abdominal discomfort with bloody mucous diarrhoea
alternating with periods of constipation or remission to
acute or fatal dysentery with fever, chill and bloody or
mucoid diarrhoea (Benenson, 1995). Dissemination
can carry the parasite to the other vital organs like liver,
where they produce focal necrosis.
In view of the zoonotic importance of amoebiasis, the
present study was designed to study the prevalence of
amoebiasis in dogs and their owners by using rapid
latex agglutination test, flotation method and direct
faecal examination.
Determination of the prevalence of E. histolytica
Collection of faecal samples: A total of 100 faecal
samples (dogs, n = 50; humans, n = 50) were examined
for amoebiasis. These samples were collected from Pet

Amoebic dysentery in dogs
75
centre, University of Veterinary and Animal Sciences, vary according to socio cultural habits, geographic
Lahore and Lahore Animal Hospital. History of dogs factors, climate and route of transmission. In the present
and their owners was regularly noted in prescribed study, an overall 11% prevalence of anti-Entamoeba
proforma. For faecal diagnosis of E. histolytica, both antibodies was detected. In dogs, a 14% faecal
direct faecal examination and flotation method were prevalence was detected, whereas in man it was 10 %.
used, as described by Soulsby (1982). Serum prevalence of dog and man was 12% and 10 %,
respectively. Many workers from different parts of the
Collection of Serum Samples: A total of 100 serum
world have reported the prevalence of anti-Entamoeba
samples (dogs, n = 50; humans, n = 50) were tested for
antibodies by using various serological techniques
amoebiasis. Under aseptic measures, 8–10 ml volumes
including latex agglutination test (LAT), e. g. Rizwan
of blood were drawn by veinipuncture with the help of
et al. (1999) have reported that 15-20% dog owners
disposable syringes and transferred slowly to screwfrom
Karachi (Pakistan) and 12 % pet owners in
capped sterile test tubes, to avoid hemolysis (Benjamin,
Tanzania (Gilli et al., 1992) had anti-Entamoeba
1985). Serum samples were then separated by first
antibodies in their serum. However, highest (35 %)
clotting the blood and then centrifugation at 3500 rpm
prevalence has been reported by Abioye (1972) in
for 5 min. All the collected serum samples were
Ibadan. This variation in the results could be due to
analyzed for specific Entamoeba antibodies by using
difference in environmental and geographical
latex agglutination test. For this purpose, the
conditions. It is interesting to note that pet keeping led
commercial test kit “Entamoeba Latex”, manufactured
to increase in the incidence of amoebiasis (Anand et al.,
by Quimica Clinica Apelicada, SA Asmota, Spain, was
1997). Further, it has been observed that if a dog owner
used.
is infected with amoebiasis, his dog is also found to be
Amoebiasis is a disease that is dispersed worldwide, infected. It may thus mean that, most probably, the dog
and its seropositivity levels widely vary in different owners could contract infection from their dog.
regions of the globe (David 1972). The prevalence rates Curiously, amoebiasis is age related as its prevalence is
Table I. Distribution of E. histolytica in humans determined by using latex aagglutination, direct fecal examination
method and flotation method
Sample No. Anti body Direct Flotation % % Fecal
tested titer fecal Seropositive
reciprocal
method
16 128 256
Serum of dog 50 1 1 2 - - 8 -
owners
Fecal of 50 4 5 10
dog owners
Total 100 1 1 2 4 5 8 10
Table II. Distribution of E. histolytica in various age groups of dogs by using latex agglutination test
Age group No. tested Reciprocation Seropositive %
(years)
Seropositive
1:16 1:128 1:256
0-2 06 1 1 16.6
3-4 15 1 1 1 3 20
5-6 16 1 1 6/23
7 and above 13 1 1 7.2
Total 50 2 2 2 6 12

76 Umair et al.
Table III. Distribution of E. histolytica in various age groups of dogs by using direct fecal and flotation method
Age groups No. Positive % Positive Positive % Positive
(years) tested fotation flotation direct direct
method method method method
0-2 06 1 16.6 0 0
3-4 15 4 26.1 3 20
5-6 16 1 6.23 1 6.23
7 and above 13 2 15.4 2 15.4
Total 50 8 16 6 12
higher in animals of younger age. Dogs between 1–3
years of age are known to be maximally susceptibility
to amoebiasis.
REFERENCES
Abioye AA and Edington GM. 1972. Prevalence of amoebiasis at
autopsy in Ibadan. Transactions of the Royal Society of
Tropical Medicine and Hygiene 5:754-763.
Anand AC, Reddy PS, Saiprasad GS, Kher SK. 1997. Does nondysenteric
intestinal amoebiasis exist? The Lancet 9045:89-
92.
Benjamin MM. 1985. Outline of Veterinary Pathology III (Ed.)
Kalyani Publishers, New Delhi. pp. 102-104.
David Botero R. 1972. Studies on the stability of the
hemagglutinating antibody of Entamoeba histolytica.
Transactions of the Royal Society of Tropical Medicine and
Hygiene 66: 517-518.
Gilli S, Petithory JC, Adoin F, Pannetier C and Scaglia M. 2001.
Asymptomatic amoebic infection: Entamoeba histolytica or
Entamoeba dispar. Bulliten of Society of Pathology Exotics
94:304-307.
Gómez Maganda T, García Carrizosa R, Torres Valadez F, Ortiz
Ramírez E, Villaseñor de la Parra C , Flores González A and
Gómez García E. 1978. Latex agglutination test in amebic
liver abscess. Revista de Gastroenterologia de Mexico 43:21-
28.
Morris MN, Powell SJ and Elsdon-Dew R. 1971. Invasive
amoebiasis: circulating antibody levels by latex agglutination
test. South African Medicine Journal 45:1206-1208.
Rizwan, QM, Syed A and Hafiz A.1999. Acute and chronic
amoebiasis. The professional 6:364-371.
Soulsby EJL. 1982. Helminths, Arthropods and Protozoa of
Domestic Animals. (Ed.). Bailliere Tindall, London, UK. Pp.
765-766.

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Enzyme nomenclature: The trivial names with the disk.
recommended by the IUPAC-IUB Commissions
should be used. At the first citation in the text of the
paper, its code number and systematic name should be
indicated.
iv

JOURNAL OF PARASITIC DISEASES
(Official organ of the Indian Society for Parasitology)
Undertaking by Authors
J P D
We, the undersigned, give an undertaking to the following effect with regard to our manuscript titled:
_____________________________________________________________________________________________
_____________________________________________________________________________________________
_____________________________________________________________________________________________
submitted for consideration to possible publication in the Journal of Parasitic Diseases.
1. Research reported was conducted within the proper animal care and according to the applicable National Laws.
2. The manuscript mentioned above has not been submitted for publication in any form to any other journal.
3. The paper, if published in the Journal of Parasitic Diseases, will become the sole copyright of the Indian Society for
Parasitology and the article(s) thereof will not be published elsewhere unless written consent is obtained from the Editor-inchief.
4. We also agree to the authorship of the manuscript in the following sequence:
Authors' names (in sequence) Signature of authors
1. _________________________________________ _______________________________________
2. _________________________________________ _______________________________________
3. _________________________________________ _______________________________________
4. _________________________________________ _______________________________________
5. _________________________________________ _______________________________________
6. _________________________________________ _______________________________________
7. _________________________________________ _______________________________________
8. _________________________________________ _______________________________________
9. _________________________________________ _______________________________________
10. _________________________________________ _______________________________________
Please note:
1. All authors are required to sign independently in the form and in the sequence given above. A photocopy of this form may
also be used.
2. No addition/deletion/or any change in the sequence of the authorship will be permissible at a later stage, without valid
reasons. If change is valid , then all authors involved should attest to the change. The decision, however, rests wth the
Editor-in-chief.
3. If the authorship is contested at any stage, the manuscript will be either returned or will not be processed for publication.
Important
In order to facilitate speedy processing of the submitted manuscript, please ensure the following:
1. Manuscripts are prepared as per the format of JPD.
2. Three copies of the manuscript and a brief covering letter.
3. Running title of not more than 60 characters including spaces.
4. Keywords (3-5 words).
5. "Undertaking by Authors", duly complete and signed.
6. Three sets of photographs (if any) with legends and captions on a separate page.
7. A CD containing exactly the hard copy version.
v

The Indian Society for Parasitology
Membership Application
Name : ____________________________________________________________________________________
Educational Qualification:______________________________________________________________________
Field of Specialization :________________________________________________________________________
Work Address:______________________________________________________________________________
__________________________________________________________________________________________
Phone.:_________________________Fax:____________________________E-mail:______________________
Mailing Address:_____________________________________________________________________________
__________________________________________________________________________________________
Phone.:_________________________Fax:____________________________E-mail:______________________
Signature of the Applicant:_____________________________________________________________________
Proposed by:______________________________ Seconded by:_____________________________________
(Member, ISP)
(Member, ISP)
__________________________________________________________________________________________
Membership Fee
India : Life Membership : Rs. 2000.00 Other Countries : US$ 400.00
(Demand draft should be drawn in favour of Secretary, The Indian Society for Parasitology, payable at Lucknow)
Membership of the Society includes subscription to the Journal of Parasitic Diseases, a biannual publication of the
ISP. Members are eligible to attend annual conferences of the ISP and avail discounts in registration fee.
Membership is governed by the Constitution and Bye Laws of the Indian Society for Parasitology.
-----------------------------------------------------------------------------------------------------------------------------------------
(For Office use Only)
Life Membership No. :____________________
Receipt No.:___________________
Signature of Secretary Signature of Treasurer
----------------------------------------------------------------------------------------------------------------------------------------
Note: Duly completed form along with draft should be mailed to Dr. J. K. Saxena Secretary, ISP, Division of
biochemistry, Central Drug Research Institute, Post Box-173, Chattar Manzil Palace, Lucknow-226001, India.
Personal cheques/cash are not accepted. There is no annual or ordinary membership. This form may be xeroxed.
vi

THE INDIAN SOCIETY FOR PARASITOLOGY
Executive Committee
President
Prof. Veena Tandon
Department of Zoology
North Hill University
Shillong
Vice-President
Dr. J. Mahanta
Regional Medical Research Centre
Dibrugarh
Vice-President
Prof. P. D. Juyal
Department of Veterinary Parasitology
Guru Angad Dev Vet. & Ani. Sci. Univ.
Ludhiana
Joint Secretary
Dr. Vas Dev
Malaria Research Center
Sonapur
Vice-President
Prof. R. Madhvi
Department of Zoology
Andhra University
Vishakapatnam
Secretary
Dr. J. K. Saxena
Division of Biochemistry
Central Drug Research Institute
Lucknow
Treasurer
Dr. L. M. Tripathi
Division of Parasitology
Lucknow
Members
Dr. Wasim Ahmed, Aligarh
Dr. P. Prakash Babu, Hyderabad
Dr. S. C. Datta, Kolkata
Dr. S. K. Ghosh, Bangalore
Dr. Neena Goyal, Lucknow
Prof. Neelima Gupta, Bareilley
Dr. B. V. Jadhav, Aurangabad
Dr. A. M. Khan, Dibrugarh
Dr. Ashwani Kumar, Goa
Prof. G. G. Mani, Visakhapatnam
Dr. S. K. Puri, Lucknow
Prof. R. Kaleysa Raj, Trivandrum
Dr. K. K. Saxena, Bareilley
Prof. Prati Pal Singh, S. A. S. Nagar
Statements and opinions expressed in the Journal of Parasitic Diseases or in the presentations
during the regular meetings of the Indian Society for Parasitology are those of the author(s), and
do not necessarily reflect the official position of the Society. The Editorial Board, the publisher
and the Society disclaim any responsibility for the accuracy of the statements made by the
contributors.
Copyright © 2008 The Indian Society for Parasitology

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