June 2008 - The Indian Society for Parasitology
June 2008 - The Indian Society for Parasitology
June 2008 - The Indian Society for Parasitology
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ISSN: 0971-7196<br />
Journal of<br />
Volume 32 Number 1 <strong>June</strong> <strong>2008</strong><br />
(Released September <strong>2008</strong>)<br />
Parasitic<br />
Diseases<br />
<strong>The</strong> Official Organ of the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
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INDIAN SOCIETY FOR PARASITOLO<br />
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Published by<br />
<strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
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Electronic version available on ISP Website<br />
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JOURNAL OF PARASITIC DISEASES<br />
(ISSN: 0971-7196)<br />
Editor-in-Chief<br />
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National Institute of Pharmaceutical<br />
Education and Research<br />
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National Institute of Pharmaceutical<br />
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Editorial Board<br />
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Prof. B. C. Harinath, Sevagram<br />
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Journal of Parasitic Diseases is published biannually by the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong> in <strong>June</strong> and December in<br />
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Biochemistry, Central Drug Research Institute, Chattar Manzil, Lucknow – 226 001, India.
JOURNAL OF PARASITIC DISEASES<br />
Volume 32, Number 1, <strong>June</strong> <strong>2008</strong><br />
<strong>The</strong> official organ of<br />
THE INDIAN SOCIETY FOR PARASITOLOGY<br />
Central Drug Research Institute, Lucknow-226 001, India.<br />
Editorial office: National Institute of Pharmaceutical Education and Research<br />
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Journal of Parasitic Diseases<br />
Copyright © <strong>2008</strong> <strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
All Rights Reserved<br />
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JOURNAL OF PARASITIC DISEASES<br />
Volume 32 Number 1 <strong>June</strong> <strong>2008</strong><br />
CONTENTS<br />
Invited review<br />
Parasitic zoonotic infections in Egypt and India: an overview. Neelima Gupta,<br />
D. K. Gupta and Said Shalaby<br />
1-9<br />
Review<br />
Dot-ELISA in the diagnosis of parasitic diseases of animals. A. Gupta, A. K. Dixit<br />
and Pooja Dixit<br />
10-14<br />
Original papers<br />
Lymphatic filariasis in Benue State, Nigeria: community diagnosis using the rapid<strong>for</strong>mat<br />
antigen immunochromatographic card test. E. A. Omudu and F. C. Oka<strong>for</strong><br />
Purification and characterization of Plasmodium berghei glutathione reductase.<br />
G. Kapoor, U. Bagai and H. S. Banyal<br />
Human sparganosis in Sri Lanka: a case report and review of the cases.<br />
R. L. Ihalamulla, H. Gunatilaka and S. D. Fernando<br />
Immunocytochemical demonstration of 5-hydroxytryptamine and localization of<br />
monoamine oxidase in Gigantocotyle explanatum and Gastrothylax crumenifer<br />
(Digenea: Paramphistomidae). S. Ghani, Z. Ghani and S. M. A. Abidi<br />
Seasonal dynamics in lungworm infections in sheep. N. Moghaddar,<br />
S. S. Shekar<strong>for</strong>oush and A. Afrahi<br />
Focus on pathogenic trematode cercariae infecting fresh water snails (Mollusca:<br />
Gastrapoda) of tribal region of southern Rajasthan, India. S. L. Choubisa<br />
On Paralueheia guptai n. gen. & n. sp. (Acanthocephala: Plagiorhynchinae Meyer,<br />
1931) from Stromateus sinensis. A. M. Saxena and Rahul Gupta<br />
Some epidemiological aspects of intestinal cestodes of sheep in a temperate climate.<br />
K. A. Tariq, M. Z. Chishti and F. Ahmad<br />
15-21<br />
22-29<br />
30-33<br />
34-41<br />
42-46<br />
47-55<br />
56-59<br />
60-63
Short communications<br />
Effect of the control of one-host cattle tick Boophilus microplus on growth and<br />
haematological parameters of calves. S. Vatsya, R. R. Kumar, C. L. Yadav and R.<br />
Garg<br />
Mode of nutrition in pathogenic trematode larvae (redia and cercaria) which infect<br />
hepatopancreas of fresh water snails (Mollusca: Gastropoda). S. L. Choubisa<br />
Amoebic dysentery in dogs and dog owners. Saleh Umair, Azhar Maqbool,<br />
Zubair Shabbir and M. D. Ahmad<br />
Instructions to Authors<br />
Undertaking by Authors<br />
Membership Application<br />
64-67<br />
68-73<br />
74-76<br />
i-iv<br />
v<br />
vi
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 1-9<br />
Invited review<br />
J P D<br />
Parasitic zoonotic infections in Egypt and India: an<br />
overview<br />
1 2 3<br />
Neelima Gupta , D. K. Gupta and Said Shalaby<br />
1<br />
Department of Animal Science, M. J. P. Rohilkhand University, Bareilly.<br />
2<br />
Department of Zoology, Bareilly College, Bareilly.<br />
3<br />
Department of Complementary Medicine, Medical Division, National Research Centre, Cairo, Egypt.<br />
Received 2 September <strong>2008</strong>; revised 8 September <strong>2008</strong>; accepted 17 September <strong>2008</strong><br />
ABSTRACT. Zoonotic infections are diseases transmitted from vertebrate animals to man, and thus<br />
compromise people's health as well as endanger their livelihoods by affecting livestock and/or other<br />
useful domestic animals. Zoonotic diseases are an important group of infectious diseases within the<br />
broader domain of veterinary public health. Changes in social, dietary and cultural mores and<br />
environmental changes have caused a scourge in zoonotic diseases in Africa and Asia continents,<br />
which World Health Organization has estimated to be the most zoonotic prone continents. Parasitic<br />
zoonotic in<strong>for</strong>mation in these continents are scattered, and the present communication attempts to<br />
review the prevailing parasitic zoonotic diseases in these continents by selecting Egypt (Africa) and<br />
India (Asia) as the two representative countries. Herein, the zoonotic infections in Egypt and India,<br />
with reference to protozoal and helminthic diseases, are overviewed, which indicate the susceptibility<br />
of the populations of these two countries to zoonotic diseases. <strong>The</strong> classification, mode of transmission<br />
and control of zoonotic infections are also discussed.<br />
Keywords: echinococcosis, filariasis, leishmaniasis, toxoplasmosis, zoonoses<br />
INTRODUCTION<br />
Zoonosis (Greek: “zoon” = animal) is an important<br />
group of infectious diseases within the broader domain<br />
of veterinary public health. Food and Agricultural<br />
Organization/World Health Organization (WHO)<br />
expert committees call it “the infectious diseases that<br />
are naturally transmitted between animals and man”.<br />
<strong>The</strong> diseases transmitted from humans to animals are<br />
sometimes called reverse zoonoses or anthroponoses<br />
(Greek: “anthropos” = man, “nosos” = disease, Noble<br />
and Noble, 1982). During the last 20 years, 70% of the<br />
emerging diseases have been found to be zoonotic in<br />
Corresponding author: Prof. Neelima Gupta, Department of<br />
Animal Science, M. J. P. Rohilkhand University, Bareilly-243<br />
006, India. E-mail:guptagrawal@rediffmail.com<br />
nature, and about 300 diseases are common to both man<br />
and animals. Human health is inextricably linked to<br />
animal health and production. <strong>The</strong> close association<br />
between animals and man can lead to serious risk to<br />
public health with severe economic consequences.<br />
Consequently, a number of communicable diseases<br />
(zoonoses) can be transmitted from animals to humans.<br />
Many of them have been reported infrequently or<br />
locally only, whereas some are relatively unimportant<br />
from both public health and economic points of view.<br />
Zoonoses affect man in a variety of ways by causing<br />
illness, delays, termination, monetary losses in the <strong>for</strong>m<br />
of cost of drugs and physician's fee, loss of wages and<br />
work efficiency, medico-legal complications and<br />
unfavorable publicity.<br />
A zoonotic agent may be a bacterium, a virus, a fungus
2 Gupta et al.<br />
or a parasite like protozoan or helminth. Some of these 7. Fish and shell fish: Heterophyes<br />
pathogens cause diseases in humans but sub-clinical<br />
heterophyes, Diphyllobothrium latum,<br />
infection in animals makes it difficult to eradicate the<br />
Capillaria phillippinesis, Clonorchis<br />
diseases caused by them. Over 75% of the new diseases<br />
sinensis, Gnathostoma spinigernum,<br />
that have affected humans over the past 10 years have<br />
Angiostrongylus cantonensis (eating<br />
been caused by pathogens originating from an animal or<br />
infected snails or crabs), Prohemistomum<br />
products of animal origin. Many of these diseases have<br />
vivax and Haplorchis pumilio.<br />
the potential to spread through various means over long<br />
distances to become global problems. A vast majority of<br />
Zoonoses has also been classified according to the<br />
them are not prioritized by health systems at national<br />
ecosystem in which they circulate (Hubalek, 2003):<br />
and international levels and are, there<strong>for</strong>e, labeled as 1. Synanthropic zoonoses: Urban (domestic)<br />
neglected diseases.<br />
cycle in which the source of infection is<br />
<strong>The</strong> emerging interdisciplinary field of conservation<br />
domestic and synathropic animals<br />
medicine, which integrates human and veterinary 2. Exoanthropic zoonoses: Sylvatic (feral and<br />
medicine as well as environmental sciences, is largely<br />
wild) cycle a natural focci outside human<br />
concerned with zoonoses. <strong>The</strong> overall concept of<br />
habitats.<br />
zoonoses is complex. It involves man, an invertebrate<br />
(usually arthropod), the causative agent and the 3. Both urban and natural cycles.<br />
environment – all <strong>for</strong>ming a biological whole (Shah, 1987).<br />
MODES OF TRANSMISSION<br />
Zoonotic diseases in Africa and Asia are of importance<br />
Zoonotic diseases may be transmitted by one of the<br />
as these continents are especially prone to parasitic<br />
following modes (Shalaby and Gupta, 1997)<br />
zoonotic diseases. Keeping this in view, the present<br />
study was undertaken to compare the zoonotic diseases 1. Inhalation<br />
prevalent in an African country, Egypt and an Asian<br />
country, India.<br />
2. Ingestion: Via meat e. g. beaf as in Taenia<br />
saginata, pork as T. solium, trichonosis<br />
CLASSIFICATION OF ZOONOSES<br />
and Balantidium coli.<br />
Zoonoses have been classified according to their 3. Contact with animals<br />
reservoir hosts:<br />
4. Arthropod vectors<br />
1. Cattle: Taenia saginata, screw worm,<br />
Babesia bovis and toxoplasmosis.<br />
2. Sheep and goat: Fasciola hepatica.<br />
3. Pigs: Balantidium coli, T. solium,<br />
Trichenella spiralis, Fasciolopsis buski<br />
and isosporiasis.<br />
4. D o g s : D i p y l i d i u m c a n i n u m ,<br />
Echinococcus granulosus, Ancylostoma<br />
caninum, toxocariasis, reservoir <strong>for</strong><br />
Leishmania donovani, L. tropica and<br />
Chagas disease,<br />
•Fleas as Diphylidium caninum,<br />
Hymenolepis nana, H. diminuta.<br />
•Mosquito-borne infections<br />
•Tick-borne disease such as<br />
babesiosis.<br />
•Sandfly-borne such as leishmaniasis.<br />
PARASITIC ZOONOTIC DISEASES<br />
Protozoal zoonoses<br />
5. Cats: Toxocara cati, toxoplasmosis and as Leishmaniasis: Three varieties of leishmaniasis have<br />
reservoir <strong>for</strong> leishmaniasis and Chagas been reported; cutaneous, mucocutaneous (Espundia)<br />
disease.<br />
and visceral (Kala Azar, Dum dum fever or ponos),<br />
which are serious, debilitating and disfiguring<br />
6. Rodents and rats: Hymenolepis nana, H. diseases. Cutaneous leishmaniasis (CL) is also known<br />
diminuta.<br />
as 'bouton d'Orient, 'Oriental sore', 'Baghdad boil',<br />
Delhi sore and 'Aleppo button'. <strong>The</strong> causative agents are
Parasitic zoonotic infections in Egypt and India<br />
3<br />
generally Leishmania tropica, L. major and L. Toxoplasmosis: Toxoplasmosis is caused by<br />
aethtiopica. <strong>The</strong> infection produces a skin ulcer which Toxoplasma gondii that is known as a potential parasite<br />
leaves an unsightly scar on healing.<br />
<strong>for</strong> man <strong>for</strong> many years but its true nature as a coccidian<br />
was discovered during 1969-70 (Smyth, 1994).<br />
Leishmaniasis of man are generally transmitted by the<br />
Domestic cats predominate as reservoir at home and in<br />
bite of female sandflies. However, the distribution and<br />
the laboratory environment. Mice, rats, hamsters,<br />
transmission is complicated by the fact that the disease<br />
guinea pigs, rodents, rabbits, dogs, sheep and cattle are<br />
is a zoonosis and there are a wide range of reservoir<br />
intermediate hosts. Man acquires infection either by<br />
hosts. CL is associated with wild rodents and dogs,<br />
direct ingestion of oocysts from a cat or by eating raw or<br />
which facilitate transmission to humans and cause<br />
undercooked meat infected with tissue cysts of<br />
zoonosis. <strong>The</strong> known reservoir hosts have been<br />
Toxoplasma sp. Cooks or butchers or persons handling<br />
reviewed by WHO (1984).<br />
raw meat are particularly at risk. Any cat, no matter how<br />
From Egypt, Morsy et al. (1995) reported that zoonotic well cared <strong>for</strong>, may be passing Toxoplasma oocysts. As<br />
CL was the most important zoonotic disease in the the disease is congenital, there<strong>for</strong>e, a pregnant woman<br />
country. <strong>The</strong> results showed that rodent population is is advised not to handle cat's faeces.<br />
more or less common in Al-Arish City (Rattus rattus<br />
In Egypt, the disease is responsible <strong>for</strong> a significant<br />
and R. norvegicus ), Bir Lehfan (Gerbillus pyramidum)<br />
percentage of abortions among inhabitants of Egyptian<br />
and Abo Oegela ( G. pyramidum , Meriones crassus and<br />
cities and rural areas . Babies may have problems of<br />
Jaculus jaculus). Phlebotomus papatassi is the main<br />
vision after being delivered normal at a later age<br />
vector being common in nearly the whole of Sinai.<br />
(Shalaby,1989; Abdelhamed, <strong>2008</strong>) .<br />
Isolates recovered from P. papatassi trapped in Sad Al-<br />
Rawafei and G. pyramidinum trapped in Abo Ogela From India, Chhabra et al. (1981) discussed the<br />
proved (isoenzyme cellulose acetate electophoresis) to epidemiological aspects and public health potential of<br />
be L. major biochemically indistinguishable from each subclinical toxoplasmosis in dairy goats with reference<br />
other. .<br />
to consumption of undercooked goat's meat and raw<br />
goat's milk. Toxoplasmosis in <strong>Indian</strong> AIDS patients has<br />
From India, Sharma et al. (1973) studied the<br />
been recognised as an opportunistic infection from<br />
epidemiological and entomological features of an<br />
India. Mittal et al. (2005) recorded the prevalence of<br />
outbreak of CL (caused by L. tropica) in Bikaner city,<br />
toxoplasmosis in <strong>Indian</strong> women of child bearing age,<br />
Rajasthan. Infection with L. major has been reported to<br />
which was due to ingestion of undercooked or<br />
be a typical zoonoses in the dry terrain of the Rajasthan<br />
uncooked meat. Sundar et al. (2007) evaluated the<br />
canal region, whereas in Bihar, most of the cases of<br />
seroprevelence of T. gondii in healthy adult population<br />
Kala Azar were caused by L. donovani (Peters et al.,<br />
of blood donors in Karnataka.<br />
1981). Mittal et al. (1991) evaluated the sensitivity and<br />
specificity of three serological tests, indirect Giardiasis: Animals considered to be sources of<br />
immunoflourescent antibody (IFA) test, enzyme- infection include rodents (rats, voles, beavers,<br />
linked immunosorbant assay (ELISA) and counter muskrats), dogs and cats and hoofed animals, beavers<br />
immunoelectrophoresis (CIEP) <strong>for</strong> the diagnosis of appear to be the animals most commonly infected<br />
<strong>Indian</strong> Kala Azar. <strong>The</strong> zoonotic reservoir of Kala Azar (Bemrick and Erlandsen, 1988). Dogs are implicated as<br />
has been identified in Bihar, where more than 1,00,000 zoonotic source of infection (Fayer et al., 2004) <strong>for</strong><br />
cases alone were reported out of 4,00,000 cases giardiasis. Although the majority of patients with<br />
recorded worldwide (Kishore and Kar, 1996). Singh chronic diarrhoea in Egypt suffer from Entamoeba<br />
(2006) estimated more than 90% of visceral histolytica infection; nevertheless, there are some cases<br />
leishmaniasis (VL) to be from India, Bangladesh, of Giardia lamblia infection being mostly of zoonotic<br />
Southern Sudan, Nepal and north east Brazil. He origin.<br />
suggested various modes of parasite transmission<br />
including vector-borne, blood transfusion, needle<br />
Malaria: Malaria is one of the world's greatest killers.<br />
sharing and sexual (person to person). Sundar et al.<br />
One of the surprising research developments has been<br />
(2007) conducted a randomized, controlled, phase 3<br />
the discovery that certain species of simian malaria are<br />
open label study comparing paromomycin with<br />
capable of infecting man and thus causing zoonosis.<br />
amphotericin B <strong>for</strong> the standard care and control of<br />
<strong>The</strong> blood <strong>for</strong>ms of Plasmodium knowlesi are known to<br />
Kala Azar in Bihar, India.<br />
be transmissible to man, though it was earlier believed
4<br />
Gupta et al.<br />
that monkey malaria was not usually infective to man. reported. Filariasis caused by W. bancrofti is most<br />
During the 1960's, the concept changed and the report common. <strong>The</strong> disease causes lymphatic obstruction<br />
that malaria from apes and monkeys can be transmitted with swollen legs or in some cases, even the whole<br />
to humans and the first proof of a simian malaria lower half of the body may be swollen . <strong>The</strong> morbid<br />
parasite causing zoonosis in man was reported by Chin changes initiated by the parasite are initially confined to<br />
et al. (1965), which involved a case of natural infection the lymphatic system and the clinical manifestations are<br />
of man by P. knowlesi, a species normally known to commonly called wuchereriasis (Filariasis). Filariae<br />
infect monkeys. Although the prevalence of malaria is occur in a wide range of tissue habitats: lymph glands,<br />
high but zoonotic cases are comparative few. From deep connective tissues, subcutaneous tissues or<br />
India, Das et al. (1996) recorded zoonoses by mesenteries. <strong>The</strong> revolting fleshy de<strong>for</strong>mities are<br />
sporozoans from West Bengal, where zoonotic malaria commonly termed elephantiasis.<br />
infection was reported in a 23 year old student who<br />
inhabited an area of gibbons which carry a malarial<br />
Strongyloidiasis: Many vertebrates, not only<br />
parasite similar to Plasmodium.<br />
mammals but also amphibians, reptiles and birds are<br />
affected by th strongyloidiasis. <strong>The</strong> available source of<br />
Sarcocystosis (Sarcosporidiosis): Sarcocystosis, the laboratory material is the faeces of farm animals<br />
obligatory heteroxenous parasite produces muscle containing eggs and larvae of the parasite. Best known<br />
cysts in intermediate hosts such as domestic herbivores species are Strongyloides stercoralis in man, cat and<br />
(cattle, sheep, pigs) and rodents. Final hosts are dog; S. fulleborni in primates other than man; S.<br />
predators such as dogs, cats, owls, snakes and man. papillosus in ruminants; S. ratti in rodents; S. ransomi<br />
Cases of human infection in muscles have been in pigs and S. westerni in equines. In man, S. stercoralis<br />
reported, probably from organisms normally found in causes strongyloidiasis, a major intestinal infection<br />
monkeys (Beaver et al., 1979).<br />
(Grove, 1989), which is widely distributed in the tropics<br />
and subtropics. <strong>The</strong> pathology of the disease has been<br />
Work on prevalence, morphology, life cycle,<br />
reviewed by Georgi (1982) and Grove (1989).<br />
transmission, pathogenesis immunology, biochemistry<br />
Strongyloidiasis, caused by S. stercoralis, is always<br />
and prophylaxis of sarcocystosis in domestic animals of<br />
found as a natural infection in dogs and cats in Egypt<br />
India was done by Juyal and Cross (1991), who<br />
and few cases of zoonosis in man have been reported.<br />
correlated the prevalence of sarcocystosis with foodborne<br />
parasitic zoonosis and stressed upon the Angiostrongylosis: <strong>The</strong> disease is caused by the<br />
emerging problems in food-borne parasitic zoonosis nematode worm Angiostrongylus that is normally<br />
and their impact on agriculture and public health. found in dogs and the intermediate hosts are land snails<br />
Helminthic zoonoses<br />
and slugs. Canine angiostrongylosis is usually a chronic<br />
condition extending over months or even years.<br />
A. Nematodes<br />
Sometimes the infection is zoonotic in humans. <strong>The</strong><br />
parasite can cause the syndrome of meningoen-<br />
Trichinoses (Trichinellosis): Trichinoses is due to cephalitis. <strong>The</strong> first record of the disease to be zoonotic<br />
Trichinella spiralis infection where the reservoirs are was from Formasa (Chatterjee, 1995). Human<br />
swine, dogs, cats and rats. It is a serious and often fatal infection occurs by ingestion of raw vegetables<br />
disease. This is essentially a 'domestic' or 'synanthropic' containing the third stage larva of the parasite or<br />
parasite and has since long been recognized to cause ingestion of tissues of improperly cooked infected<br />
zoonosis and is transmitted to man by the ingestion of intermediate hosts (snails) and carrier or paratenic host<br />
infected pork or sausages. <strong>The</strong> disease often remains (crabs, prawns, pigs). Infection may also occur by<br />
undetected during life and is thus an important health drinking contaminated water containing the infective<br />
problem.<br />
larvae. <strong>The</strong> global dispersal of peridomestic zoonoses,<br />
angiostrongylosis caused by A. cantonensis, the<br />
Filariasis: <strong>The</strong> disease is caused by the filarial worm,<br />
lungworm of rats and eosinophilic meningitis in<br />
Wuchereria bancrofti which is a widely distributed<br />
humans due to the worm has also been reported (Kliks<br />
parasite. Filarial parasites are probably the most<br />
and Palumbo, 1992; Mahajan et al., 1992).<br />
important group of helminthic infection in humans but<br />
they are of marginal concern to the veterinarian since Ascariasis: Ascaris lumbricoides, a parasite of man<br />
domestic animals are of little significance in their and pigs is cosmopolitan in distribution. <strong>The</strong> infection<br />
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<br />
5<br />
may suffer from ascariasis. <strong>The</strong> infection results in the first record of heterophyid infection was made by<br />
malnutrition and retardation in growth of children, Mahanta et al. (1995) from Dibrugarh, Assam. Only<br />
pneumonitis, asthma, diarrhoea, nausea, abdominal two cases were reported to have passed eggs in their<br />
pain and anorexia. In some cases, the infection may stools and due to the paucity of availability of these<br />
become zoonotic. Ascariasis and visceral larval parasites from India, their pathogenecity is practically<br />
migrants are always found in rural Egypt or even in untouched.<br />
urban primary school children who are fond of playing<br />
with dogs.<br />
Black Spot Disease: <strong>The</strong> disease is caused by the<br />
metacercaria of trematodes. From India, the disease has<br />
B. Trematodes been reported in Catla catla, Cirrhina mrigala, Labeo<br />
rohita and Schizothorax richardsonii due to<br />
Fascioliasis: Fascioliasis commonly occurs in sheep,<br />
metacercarial infection of Neascus and Diplostomum<br />
goats and cattle although a wide range of other hosts<br />
species (Malhotra and Bannerjee, 1989). <strong>The</strong> black<br />
have been reported. Man is usually not considered to be<br />
spots represent encysted metacercaria surrounded by<br />
a host of Fasciola hepatica but the eating of water cress<br />
th<br />
reaction pigmentation and outer cysts of 1/200–1/7 of<br />
appears to be a common source of human infection<br />
an inch in diameter surround the parasite cysts, the cysts<br />
(Marsden and Warren, 1984). Although the usual site<br />
thus consist of an outer thick cellular layer (of host<br />
of infection is the liver but in aberrant hosts like man,<br />
origin) and an inner thin non-cellular layer (of parasite<br />
the lung may be involved. Human fascioliasis infection<br />
origin). <strong>The</strong> intense black colour of the cysts is attained<br />
occurs after swallowing encysted matacercariae on<br />
due to the presence of melanocytes in the outer layer.<br />
unwashed green lettuce. <strong>The</strong> irrigation of this plant by<br />
Not only can the older cysts rupture to enable<br />
contaminated water with cercaria from water drains<br />
reinfection/fresh infection in other fish, but the<br />
containing the intermediate host snail and exposed to<br />
metacercaria may also be directly transmitted by<br />
faeces of infected cattle and sheep helps in propagation<br />
drinking of infected water, and consumption of such<br />
of infection (Beaver et al.,1984; Shalaby and Gupta,<br />
uncooked infected fish can cause infection in man. <strong>The</strong><br />
2000).<br />
parasite can be directly transmitted during extensive<br />
Schistosomiasis: Schistosomes are the blood flukes of parasitaemia (Bannerjee and Malhotra, 1989) and can<br />
mammals and birds. It commonly lives in the veins of also be zoonotically transmitted (Neascus vetesai and<br />
the mesenteries of the colon of man, wild rodents and N. channi) to homeotherms (Malhotra and Chatterjee,<br />
primates, and causes a debilitating disease commonly 1996). <strong>The</strong> zoonotic potential and environmental<br />
known as schistosomiasis. Schistosomiasis is perhaps interactions in fish parasite systems was discussed by<br />
the most important disease of helminthic origin in the Malhotra and Chatterjee (1996) and the dogma of black<br />
world today. <strong>The</strong> disease is caused by the trematode spot disease in Garhwal Himalayas and freshwater<br />
parasite Schistosoma, which parasitises all domestic ecosystems of gangetic plains was also discussed<br />
mammals mainly sheep and cattle. Water snails are (Malhotra et al., 2002) but its zoonotic signifance has<br />
important in the transmission of the disease. From not been pointed out by the authors.<br />
India, the possibility of schistosomiasis in man by S.<br />
incognitum (normal host Macaca mulatta) has been<br />
C. Cestodes<br />
discussed by Ahluwalia (1972).<br />
Echinococcosis (Hydatid disease): Echinococcus<br />
granulosus, commonly called as the hydatid worm,<br />
Heterophyiasis: Heterophyiasis infection caused by<br />
parasitises members of the canine family and foxes as<br />
Heterophyes heterophyes, an intestinal parasite of dogs,<br />
adults but humans can aquire zoonotic infection with<br />
cats, fox, humans is commonly found in Asia and Egypt<br />
hydatid larvae. E. granulosus causes one of the most<br />
and has been introduced into Hawaii. Reports also exist<br />
serious larval tapeworm infections in man. <strong>The</strong> larva of<br />
from Palestine and Far East. Eating of raw or uncooked<br />
this very dangerous species is the cause of alveolar<br />
fish causes heterophyiasis in humans. <strong>The</strong> disease is not<br />
(multilocular) hydatid diseases in man, has variable<br />
common in India. Gill (1972) recorded the parasite in<br />
clinical manifestations making diagnosis complex and<br />
two out of 88 cats sampled at All India Institute of<br />
difficult. Normal hosts <strong>for</strong> the adult parasite are dogs in<br />
Medical Sciences, New Delhi, although helminth<br />
which hundreds of worms may occur in small intestine.<br />
parasites were present in 84 samples. <strong>The</strong> first record of<br />
Wolves and jackals also harbour the adult worm and in<br />
H. dispar from Madras at an infection rate of 96% was<br />
some areas, foxes are probably affected. Human<br />
reported by Rajavelu and Raja (1988). From humans,<br />
echinococcosis (hydatidosis) is commonly the result of
6<br />
Gupta et al.<br />
intimate contact with dogs; however, other unique cases of zoonosis by mites, O. bacoti (usual hosts rats)<br />
ethnic customs favour infection. Animals primarily in two laboratory personnel and six veterinary students<br />
involved are dogs, sheep, cattle, swine, rodents and in Haryana were recorded by Ram et al. (1986).<br />
deer. Man becomes infected by eating faecal eggs,<br />
contaminated water or food stuffs or from handling<br />
CONTROL<br />
infected dogs especially on farms resulting in the Intervention and control of zoonoses requires concerted<br />
ingestion of eggs.<br />
ef<strong>for</strong>ts between the veterinary and human health sectors<br />
Echinococcosis in Egypt causes cystic disease in liver,<br />
as it requires both men and animals. WHO has taken the<br />
lungs and long bones. <strong>The</strong> definitive host is carnivorous<br />
lead in bringing together international and national<br />
animals, especially dogs. <strong>The</strong> eggs are passed in dog's<br />
organisations to deal with the problems posed by both<br />
faeces and are accidentally swallowed by humans.<br />
emerging and endemic zoonoses. <strong>The</strong> control methods<br />
From India, Parija (1991) employed serodiagnostic<br />
may target:<br />
tests to detect circulating hydatid antigen (Cag) in the<br />
serum by ELISA and other assays in humans.<br />
1. <strong>The</strong> human host (treatment, immunization).<br />
2. Insect, tick or mite vectors (repellents, insect<br />
Hymenolepiasis: Hymenolepiasis caused by sterilization, insect traps, insecticidal sprays,<br />
Hymenolepis diminuta is also frequently seen in rural<br />
bed nets, naturalistic methods eg. drainage,<br />
areas of Egypt where bread, cereals or any stored food is<br />
vegetation clearing, biological control<br />
left uncovered, being exposed to reservoirs of this<br />
methods using bacteria or viruses pathogenic<br />
worm, rodents, fleas, beetles and cockroaches. Usually,<br />
<strong>for</strong> the vector).<br />
swallowing of the infected arthropod causes this<br />
infection. <strong>The</strong> definitive hosts of the parasites are rats 3. Animal reservoir (treatment, elimination,<br />
and other rodents and are occasionally found in other<br />
immunization).<br />
mammals including man.<br />
4. Deep freezing (-15°C <strong>for</strong> 20 days be<strong>for</strong>e<br />
Dipylidiasis: Dipylidiasis is caused by the cestode<br />
distribution of meat).<br />
parasite, Dipylidium caninum. It is the commonest<br />
tapeworm genus of domestic dog and cat and rare cases<br />
Parasitic zoonotic infections: Egypt and India<br />
of human zoonoses have been reported. Children are Egypt<br />
probably infected by having their faces licked by a dog<br />
just after the dog has nipped a flea, as the fleas are the In Egypt, studies on parasites transmitted to man<br />
intermediate host of the parasite. <strong>The</strong> infection may also through fish have been conducted by Shalaby and his<br />
result by accidental ingestion of infected fleas while collegues since 1980 till date. Many parasites of<br />
fondling cats and dogs.<br />
zoonotic importance have been reported: Aliptrema<br />
tilapia was recorded as a new parasite of zoonotic<br />
From India, a case in a child from Calcutta has been importance occurring by eating improperly cooked<br />
reported from which two adult worms were recovered Nile fishes (Shalaby, 1993). Phagicola ornamentata<br />
(Chatterjee, 1995). A unique case of dipylidiasis and Pharyngostomum flapi were recorded as new<br />
infection with D. canimum in a four year old boy is said species (Shalaby Esposito et al., 1993). On the other<br />
to have been acquired through pet cats as a zoonotic hand, Prosostephanus industrius, Procerovum<br />
infection (Gadre et al., 1993).<br />
calderoni and Moedlingeria amphorae<strong>for</strong>mis were<br />
MITES<br />
recorded <strong>for</strong> the first time from Egypt (Shalaby et al.,<br />
1993). Phagicola ornamentata and Pharyngeostomum<br />
Ornithonyssus bacoti is the tropical rat mite. It is mostly flapi were reported as new species resulting due to<br />
found on rats but also attacks mites and even man and infection from Tilapia nilotica, whereas P. calderoni<br />
may give a painful bite. It also acts as a vector of and M. amphorae<strong>for</strong>mis were again recorded from<br />
Litomosoides carinii, a filarial nematode parasitic in Egypt <strong>for</strong> the first time. Lake Quarun (Fayoum<br />
rodents. <strong>The</strong>y serve in the transmission of certain province) fishes were investigated where Mugil<br />
rickettsial and viral diseases. <strong>The</strong> mite plays a minor cephalus and M. capito were found infested with both<br />
role in transmission among reservoir hosts and Heterophyidae and Diplostomatidae encysted<br />
occassionally to man of endemic typhus, rickettsial metacercariae in their muscles. Solea vulgaris showed<br />
pox, Q fever, tularimia, plague and viruses. Sporadic diplostomidae encysted metacercariae in their muscles.
Parasitic zoonotic infections in Egypt and India<br />
7<br />
Prohemistominae encysted metacercariae were again Twenty-one contributions were presented on<br />
encountered <strong>for</strong> the first time from M. cephalus. Worms “Zoonoses” in the Symposium of <strong>Indian</strong> Association<br />
caused acute enteritis in case of heavy infestation <strong>for</strong> Advancement of Veterinary <strong>Parasitology</strong> in 1987,<br />
(Shalaby et al., 1996). Shalaby et al. (2002) reported and 24 papers were presented in a Seminar on Zoonoses<br />
that human fish-borne trematode helminthoses caused in 1989, which discussed the diseases transmissible<br />
gastrointestinal symptoms such as indigestion, from animals to man including protozoan and helminth<br />
diarrhoea and abdominal pain including heart problems diseases. Dutta (1991) and Dutta and Dutta (1991)<br />
ranging from arrhythmia to ventricular premature beats presented a background in<strong>for</strong>mation on zoonoses in a<br />
as well. Sometimes obstructive jaundice, complicated seminar on Zoonoses. <strong>The</strong> Global Meet on Parasitic<br />
by sepsis and disseminated intravascular Diseases in 1996 also focussed on Zoonoses in a<br />
coagulopathy–like syndrome was evident. Shalaby et Symposium. Zoonoses maybe said to consist of five<br />
al. (2006) reported Heterophyes heterophyes, H. h. components: parasites, maintenance/reservoir host,<br />
nocens, H. katsuradai , H. dispar and H. aequalis as intermediate host and vector, susceptible/potential host<br />
fish-borne helminth zoonoses and stated that diarrhoea and environment, and each of these components may<br />
was the main clinical manifestation. interact with the other (Fig. 1).<br />
India<br />
Zoonotic diseases have been a favoured topic <strong>for</strong><br />
organising symposia in India. During the Asian<br />
Congress of <strong>Parasitology</strong> held at Bombay in 1978,<br />
zoonoses occupied an important place <strong>for</strong> deliberations.<br />
Food-borne parasitic zoonoses have a major impact on<br />
the health and economy in developing countries in the<br />
tropics and sub-tropics. Throughout India, problems of<br />
food-borne parasitic zoonoses differ due to varied food<br />
habits. Bhatia (1991, 1992) reviewed the status of<br />
various food-borne parasitic diseases in India and put<br />
Intermediate<br />
host and vector<br />
Susceptible/<br />
potential host<br />
ZOONOSES<br />
Maintenance/<br />
Reservoir host<br />
Environment<br />
Parasites<br />
Fig. 1. Components of zoonoses and their interaction
8<br />
Gupta et al.<br />
<strong>for</strong>th its impact on the health and economy in<br />
developing countries and observed variations in foodborne<br />
parasitic zoonoses due to varied food habits,<br />
unhygienic living conditions, lack of education, poor<br />
personal hygiene, poverty and occupation.<br />
<strong>The</strong> comparative reports on zoonoses in Egypt and<br />
India presented herein bring <strong>for</strong>th some of the important<br />
parasitic zoonotic diseases of the two countries, which<br />
report in<strong>for</strong>mation on the incidence, occurrence,<br />
clinical manifestations and diagnostic aids. It is<br />
envisaged that future studies will stimulate scientists<br />
<strong>for</strong> further research in this area to establish parasitic<br />
zoonotic disease potential <strong>for</strong> each disease.<br />
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24 .<br />
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Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 10-14<br />
Review<br />
J P D<br />
Dot-ELISA in the diagnosis of parasitic diseases of<br />
animals<br />
1 1 2<br />
A. Gupta , A. K. Dixit and Pooja Dixit<br />
1<br />
Department of <strong>Parasitology</strong>, College of Vetrinary Science and Animal Husbandry, Jabalpur.<br />
2<br />
Department of Clinical Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Rewa.<br />
Received 18 August <strong>2008</strong>; accepted 24 September <strong>2008</strong><br />
ABSTRACT. Traditional serological tests such as indirect haemagglutination, complement fixation,<br />
counter immunoelectrophoresis and immunofluorescence are tedious and difficult to standardize,<br />
conduct and interpret. In the last decade, enzyme-linked immunosorbent assay (ELISA) has been<br />
developed to detect a large number of parasitic diseases. However, this assay also has a number of<br />
drawbacks. In an ef<strong>for</strong>t to develop a relatively more simple to per<strong>for</strong>m and inexpensive to use assay,<br />
ELISA was modified to Dot-ELISA. <strong>The</strong> Dot-ELISA has been widely accepted as a rapid and versatile<br />
assay <strong>for</strong> the detection of protozoan and metazoan diseases in humans and livestock.<br />
Keywords: babesiosis, Dot-ELISA, fasciolosis, haemonchosis, serodiagnosis<br />
INTRODUCTION<br />
<strong>The</strong> Dot-ELISA <strong>for</strong> the demonstrartion of the parasite<br />
specific antibodies depends upon the principle that<br />
when the test serum is layered on the nitrocellulose<br />
membrane bound with the parasitic antigen, the specific<br />
antibodies if present in the serum, will bind to the<br />
corresponding antigen dot. <strong>The</strong> antigen detection Dot-<br />
ELISA may be conducted in two ways. Specimens<br />
Corresponding author: Dr. A. K. Dixit, Department of<br />
<strong>Parasitology</strong>, College of Vetrinary Science And Animal<br />
Husbandry, Jabalpur-482 001, India.<br />
E-mail: alokdixit7@ yahoo.com<br />
<strong>The</strong> Dot-enzyme-linked immunosorbent assay (Dot-<br />
ELISA) is the most frequently used assay to<br />
demonstrate either serum antibodies or antigens in the<br />
diagnosis of parasitic diseases. <strong>The</strong> Dot-ELISA has<br />
become increasingly popular as a field assay <strong>for</strong> the<br />
diagnosis of many of the parasitic diseases including<br />
fascioliasis, toxocarosis, dirofiloriosis, haemonchosis,<br />
trichinellosis, leishmaniosis and babesiosis.<br />
suspected of containing parasite antigens can be dotted<br />
in minute volumes directly on to the nitrocellulose (NC)<br />
paper and allowed to dry. Alternatively, antigen in test<br />
samples may be captured using a sandwich Dot-ELISA<br />
that is capture anti body (monoclonal or polyclonal) is<br />
dotted on to the filter paper. <strong>The</strong> test sample is then<br />
incubated with antibody filter discs which specifically<br />
capture parasite antigen. After washing to remove<br />
unbound antigen and extraneous material, a second<br />
antibody specific <strong>for</strong> antigen is added, effecting a<br />
sandwich. After incubation with antigen specific<br />
antibody and/ or enzyme conjugated anti-antibody, the<br />
addition of a precipitable, chromogenic substrate<br />
causes the <strong>for</strong>mation of a coloured dot on the solid<br />
phase which is visually red.<br />
Advantages of Dot-ELISA<br />
•It is a simple and rapid assay and can be per<strong>for</strong>med<br />
in the field or poorly equipped laboratories.<br />
•It doesn't require much instrumentation and<br />
results can be observed visually by naked eye.
Dot-ELISA and diagnosis of parasitic diseases<br />
11<br />
•Require minimum basic training to the locally<br />
available technical staff.<br />
paper. Antibodies were detected in experimentally<br />
infected sheep as early as 4 weeks post infection (WPI).<br />
•It is easy to per<strong>for</strong>m and hence Dot-ELISA is a <strong>The</strong> sensitivity of the most serological assays is<br />
frequently evaluated assay <strong>for</strong> use in the field satisfactory with crude antigens but specificity remains<br />
(Parija, 1998).<br />
hampered due to cross reactivity with other parasites<br />
infecting ruminants. Ef<strong>for</strong>ts are there<strong>for</strong>e being made to<br />
•Multiple infections can be diagnosed by the use of evolve a putative antigen specific <strong>for</strong> serodiagnosis of<br />
a single Dot-ELISA strip known as multiple Dot- fasciolosis. Enzymatically active Cathepsin L-cysteine<br />
ELISA.<br />
proteinase from F. gigantica was isolated from adult<br />
Limitations of Dot-ELISA<br />
fluke E/S material and a Dot-ELISA assay utilizing<br />
dipsticks (dipstick-ELISA) was standerdidized <strong>for</strong> its<br />
•High cost of the assay restricts it wider use.<br />
application in field. <strong>The</strong> test evidenced positive<br />
reaction at 4 WPI with weekly pooled sera from<br />
•Non-availability of reagents as well as tests experimentally infected sheep (Dixit et al., 2002).<br />
locally so need to import these from the over- <strong>The</strong>se results highlighted <strong>for</strong> the first time the<br />
seas.<br />
possibilities and advantages of using pure antigen <strong>for</strong><br />
the diagnosis of experimental F. gigantica infection.<br />
•Short shelf-lives of the perishable thermo-labile<br />
Weekly pooled sera of infected buffaloes when<br />
enzyme-based reagents.<br />
subjected to dipstick-ELISA revealed antibodies at 2<br />
•It is affected by high environmental temperature<br />
WPI (Dixit et al., 2004). Further, no cross reactivity<br />
and requires proper refrigeration facilities in<br />
was observed with weekly pooled sera of buffaloes and<br />
laboratories.<br />
goats experimentally infected with Paramphistomum<br />
epiclitum (Dixit et al., 2003).<br />
Helminthic diseases<br />
Cathepsin L-cysteine proteinase from F. gigantica was<br />
Fasciolosis: In tropics, fasciolosis mainly caused by also evaluated <strong>for</strong> its potential in the early prepatent<br />
Fasciola gigantica has been a continuous constraint on detection in bovine calves. F. gigantica infection could<br />
growth, productivity and reproduction of ruminants. be detected 4 WPI using an ELISA, dipstick ELISA and<br />
<strong>The</strong> diagnosis of fasciolosis has been mainly relied Western blotting with 100% sensitivity. Preliminary<br />
upon the detection of eggs in the feces of infected studies showed that F. gigantica cathepsin L-cysteine<br />
animals. However detection of relatively small number proteinase does not cross-react with P. epiclitum,<br />
of eggs, in comparatively large amount of feces, has Gigantocotyle explanatum and hydatid cyst antigens<br />
been encountered as major bottle-neck in the diagnosis (Sriveny et al., 2006).<br />
with these methods and actually positive animals are<br />
reported false negative. Furthermore, the coproscopic<br />
To test the diagnostic potentiality of the somatic<br />
examination of eggs is impractical solution, when<br />
antigens, ELISA and dot ELISA was standardized using<br />
flukes are yet to mature as adult to discharge eggs,<br />
the sera from experimentally non-infected (group A)<br />
during prepatent period of disease.<br />
and infected (group B) animals. Further, the sensitivity<br />
and the specificity of the assays were evaluated<br />
An alternative answer to overcome above deficiencies employing the field sera from animals of different<br />
associated with coproscopic diagnosis rests in detection parasitic load viz., F. gigantica positive (group C), F.<br />
of anti-Fasciola antibodies and /or circulating antigens gigantica and Gastrothylax crumenifer positive (group<br />
by immunological methods. Immunodiagnosis is D), F. gigantica and G. explanatum positive (group E),<br />
crucial in the control of fasciolosis, as it can detect an a group of sera without F. gigantica but other trematode<br />
early prepatent infection leading to the timely infection (group F), only G. crumenifer positive (group<br />
chemotherapeutic intervention and also preventing the G), only G. explanatum positive (group H), G.<br />
contamination of water bodies with the parasite eggs crumenifer and G. explanatum positive (group I), and<br />
(Dixit et al., <strong>2008</strong>). In an ef<strong>for</strong>t to develop a more rapid PM negative (group J) collected from slaughterhouses<br />
and economical diagnostic test <strong>for</strong> fasciolosis, of Bareilly (Uttar Pradesh, India) and Patna (Bihar,<br />
Zimmerman et al. (1985) prepared a crude antigen of India). In plate ELISA, the sensitivity of the antigen and<br />
excretory/ secretory (E/S) preparation of Fasciola the assay was 75.75%, whereas the specificity was<br />
hepatica and adsorbed the mixture on the nitrocellulose 97%, 95%, and 98%, respectively, against G.
12 Gupta et al.<br />
crumenifer, G. explanatum, and mixed infection of G. natural sheep sera having H. contortus infection, 60%<br />
crumenifer and G. explanatum, respectively. In the case sera samples showed solid dot <strong>for</strong>mation whereas in F-1<br />
of Dot-ELISA the sensitivity was 86.5% and specificity fraction 75% of the sera samples showed solid dot<br />
was 92.3%, 94.7%, and 90%, respectively, against G. indicating purified fraction was a more potent antigen.<br />
crumenifer, G. explanatum, and mixed infection of G. Crude E/S and F-1 were also fractionated through SDScrumenifer<br />
and G. explanatum, respectively (Kumar et PAGE. ES antigen revealed polypeptides in the range of<br />
al., <strong>2008</strong>).<br />
10–200 kDa of which 26, 32, 60 and 120 kDa were<br />
found more prominent. F-1 fraction on SDS-PAGE<br />
Toxocarosis: Matsumura et al. (1987) conducted a<br />
analysis revealed only four polypeptides of 26, 32, 60,<br />
Dot-ELISA <strong>for</strong> detecting antibodies against Toxocara<br />
and 120 of which 60 and 120 kDa were found to be most<br />
canis infection in dogs. All assay steps were per<strong>for</strong>med<br />
prominent. Results indicate that the purified fraction of<br />
at room temperature. <strong>The</strong> well-defined blue spots<br />
ES antigen may be utilized <strong>for</strong> early diagnosis of<br />
<strong>for</strong>med on NC paper were evaluated by both visual<br />
haemonchosis. (Prasad et al., <strong>2008</strong>)<br />
observation and densitometric reading. <strong>The</strong> assay was<br />
carried out within 1.5 h.<br />
Trichinellosis: It is a parasitic disease of public health<br />
importance caused by the nematode Trichinella<br />
Dirofilariosis: Matsumura et al. (1988) conducted a<br />
spiralis. Most infections in domestic and wild animals<br />
Dot-ELISA <strong>for</strong> the detection of antibodies against<br />
go unnoticed. Microscopical examination of muscle<br />
Dirofilaria immitis in dogs. Dirofilarial antigens<br />
biopsy sample may confirm but not necessarily rule out<br />
prepared were directly bound on NC paper set into a<br />
trichinellosis.<br />
microfiltration apparatus to ensure their uni<strong>for</strong>mity. Of<br />
the 23 infected dogs tested, 21 were determined A Dot-ELISA using antigens purified by monoclonal<br />
positive by visual observation of brown-colored spots antibody affinity chromatography was developed <strong>for</strong><br />
on NC paper. <strong>The</strong> positive and negative readings were detecting T. spiralis infection in swine. (Su and<br />
further confirmed by using a densitometer. Sera from Prestwood, 1991).<strong>The</strong> assay was as sensitive as an<br />
15 non-infected and 17 other parasite-infected dogs ELISA (using E/S products as antigen) and western blot<br />
were all negative, i.e. no false positive readings and analysis and nearly as specific as western blot. <strong>The</strong> Dotcross-reactivities<br />
were found using this technique. ELISA detected all of 20 low infections (0.08-4.74<br />
Also, a large number of samples could be assayed larvae/gram of diaphragm), most of them by 5–6 WPI.<br />
simultaneously within 1.5 h. Similar results were obtained by Aguilar et al. (2000).<br />
Sera from A. suum infected swine were negative to E/S<br />
Haemonchosis: Detection of infection during<br />
antigen of T. spiralis when analyzed by Dot-ELISA and<br />
prepatency, is of greater importance from the clinical<br />
western blot. <strong>The</strong> improved specificity was achieved by<br />
point of view, there<strong>for</strong>e a simplified, field oriented Dotemploying<br />
species-specific denatured antigens. More<br />
ELISA has been developed <strong>for</strong> the detection of<br />
importantly, the Dot-ELISA was much simpler to<br />
Haemonchus contortus soluble antigen in goat /sheep<br />
per<strong>for</strong>m than western blot analysis.<br />
sera (Sood et al., 1996). Dot-ELISA per<strong>for</strong>med with<br />
immunoaffinity purified somatic antigen could detect Protozoan diseases<br />
infection as early as 1 WPI during pre-patency. Further,<br />
Leishmaniosis: It is a chronic, severe protozoal disease<br />
SDS-PAGE analysis of immunoaffinity purified<br />
of man, dogs and certain rodents characterized by<br />
antigen revealed four polypeptides (26, 32, 60 and 120<br />
cutaneous or muco-cutaneous leisions. <strong>The</strong> most<br />
kDa) which may be utilized <strong>for</strong> the detection of H.<br />
reliable diagnostic test <strong>for</strong> the canine leishmaniosis is<br />
contortus infection in sheep or <strong>for</strong> immunoprophylaxis<br />
direct observations of the amastigotes in macrophages<br />
(Prasad et al., 2007).<br />
in bone marrow or lymph nodes smears. <strong>The</strong><br />
E/S protein of H. contortus was also purified through disadvantage of these methods is that it is sometimes<br />
immunoaffinity chromatography and Dot-ELISA was impossible to detect the parasite in infected animals<br />
per<strong>for</strong>med with crude ES antigen as well as especially in lymph node smears.<br />
immunoaffinity purified fraction (F-1) with<br />
A comparative study was done <strong>for</strong> the diagnosis of<br />
experimental and natural sera of sheep infected with H.<br />
leishmaniosis by Dot-ELISA and blinded bone marrow<br />
contortus. Solid dot <strong>for</strong>mation took place with four day,<br />
aspirate (Reynaldo et al. 1995). Using visualization of<br />
1, 2 and 3 wpi sera. Dot <strong>for</strong>mation did not take place<br />
amastigotes on bone marrow examination as a priori<br />
with negative control serum and uninfected control<br />
evidence of infection, the Dot-ELISA was found to be<br />
animal serum. When crude E/S antigens was reacted to
Dot-ELISA and diagnosis of parasitic diseases<br />
13<br />
highly sensitive(97%) and specific (100%). In contrast, In addition, the Dot-ELISA may be configured to detect<br />
the physical evaluation had remarkably low sensitivity antibodies or parasite antigen in either micro-titer plates<br />
and specificity. <strong>The</strong> Dot-ELISAis an excellent assay <strong>for</strong> <strong>for</strong> large-batch testing or with dipsticks <strong>for</strong> small<br />
detection of the canine reservoir of Leishmania, Thus it numbers of determinations. <strong>The</strong> facilities <strong>for</strong><br />
may help to control this debilitating illness by indigenous production and distribution of enzyme<br />
facilitating selective canine elimination.<br />
labeled reagents by a central laboratory and easy<br />
availability of nitrocellulose membrane <strong>for</strong> coating the<br />
A Dot-ELISA was developed <strong>for</strong> diagnosing visceral<br />
proteins may reduce the cost of the assay to some extent<br />
leishmaniosis in dogs infected with Leishmania<br />
and make it a popular assay.<br />
infantum (Vercamme et al., 1998). <strong>The</strong> test procedure<br />
lasted only 30 min. Distinction between Leishmania REFERENCES<br />
positive and Leishmania negative sera was complete at Aguilar FBR, Bautista-Garfias CR, Rojas J, de Nova ME,<br />
a dilution of 1/320, hence there was no cross-reactivity, Rodríguez O I and Martinez-Gómez F. 2000. Experimental<br />
not even with sera from dogs with trypanosomal or swine Trichinellosis: Use of Dot-ELISA and western blot<br />
babesial infections. <strong>The</strong>re<strong>for</strong>e, it is concluded that this with excretion/secretion antigens (ES) from infective larvae<br />
to detect anti-Trichinella spiralis antibodies. Rev Latinoam<br />
test has a very high sensitivity and specificity <strong>for</strong> the<br />
Microbiol 42:57-62<br />
detection of anti-Leishmania antibodies in the dog. <strong>The</strong><br />
Dot-ELISA was significantly positive correlated with Camus E and Montenegro James S. 1994. Bovine anaplasmosis<br />
and babesiosis in the Lesser antilles: risk assessment of an<br />
the direct agglutination test (DAT), the indirect<br />
unstable epidemiologic situation. Vet Res 25:313-317.<br />
immunofluorescence test (IFAT), and the slide-ELISA.<br />
Similarly, Dot-ELISA using protein A-peroxidase was Dixit AK, Yadav SC, Saini M and Sharma RL. 2003. Purification<br />
evaluated as a diagnostic test <strong>for</strong> canine leishmaniasis<br />
and characterization of 28 kDa cysteine proteinase <strong>for</strong><br />
immunodiagnosis of tropical fasciolosis. J Vet Parasitol<br />
(Fisa et al., 1997). <strong>The</strong> test results were in agreement<br />
17:5-9.<br />
with parasitologic diagnosis and indirect<br />
immunofluorescence assay results.<br />
Dixit AK, Yadav SC and Sharma RL. 2002. 28 kDa Fasciola<br />
gigantica cysteine proteinase in the diagnosis of prepatent<br />
Babesiosis: It is a tick transmitted protozoan disease of ovine fasciolosis. Vet Parasitol 109:233-247.<br />
wild and domestic animals. Although microscopic Dixit AK, Yadav SC and Sharma RL. 2004. Kinetics of antibody<br />
detection of Babesia parasites is the most efficient and response using 28 kDa Fasciola gigantica cysteine<br />
cost effective method <strong>for</strong> the diagnosis of acute proteinase as antigen. Trop Anim Health Prod 36:49-54.<br />
babesiosis, the detection of specific antibody is Dixit AK, Dixit Pooja and Sharma RL. <strong>2008</strong>.<br />
recommended <strong>for</strong> epidemiological surveillance and the Immunodiagnostic/ protective role of Cathepsin L cysteine<br />
assessment of enzootic stability.<br />
proteinase secreted by Fasciola species. Vet Parasitol 154:<br />
177-184.<br />
A reagent conservative Dot-ELISA was developed<br />
Fisa R, Gallego M, Riera C, Aisa MJ, Valls D, Serra T, de<br />
primarily <strong>for</strong> canine babesiosis caused by Babesia<br />
Colmenares M, Castillejo S and Portus M. 1997. Serologic<br />
canis. Strong agreement was observed between Dot- diagnosis of canine leishmaniasis by Dot-ELISA. J Vet Diagn<br />
ELISA and the conventionally used IFAT Invest 9:50-55.<br />
(Wanduragala et al., 1987). Dot-ELISA has also been<br />
Kumar N, Ghosh S and Gupta SC. <strong>2008</strong>.Early detection of<br />
configured <strong>for</strong> the simultaneous detection of antibodies<br />
Fasciola gigantica infection in buffaloes by enzyme linked<br />
to Babesia bovis, Babesia bigemina and Anaplasma immunosorbent assay and dot enzyme linked immunosorbent<br />
marginale antibodies (Montenegro James et al., 1992). assay. Parasitol Res (in press).<br />
<strong>The</strong> multidot ELISA is especially suited <strong>for</strong> sero-<br />
Matsumura K, Kazuta Y, Endo R, Tanaka K, Inoue T and<br />
epidemiological studies in regions where all three Matsuda H. 1988. A rapid microassay <strong>for</strong> dirofilarial<br />
haemo-parasites are endemic (Camus and Montenegro antibodies by using antigen-coated nitrocellulose paper. Vet<br />
James, 1994). Mattioli et al. (1997) used the multiDot- Parasitol 27 :337-44.<br />
ELISA to survey the seasonal prevalence of tick Matsumura K, Kazuta Y, Endo R, Tanaka K, Inoue T and<br />
transmitted haemoparasites in N'Dama cattle in West Matsuda H. 1987. A rapid detection of toxocaral antibodies<br />
Africa.<br />
by dot enzyme-linked immunosorbent assay (Dot-ELISA) in<br />
CONCLUSIONS<br />
dogs. Zentralbl Baktriol Mikrobiol Hyg 266:580-585.<br />
Mattioli RC, Janneh L, Corr N, Faye JA, Pandey US and Verhulet<br />
<strong>The</strong> Dot-ELISA is rapid, easy to per<strong>for</strong>m and interpret A. 1997 .Seasonal prevalence of ticks and tick transmitted<br />
reagent conservative, cost effective and field portable. haemoparasites in traditionally managed N'Dama cattle with
14 Gupta et al.<br />
reference to strategic tick control in the Gambia. Med Vet Dot-ELISA <strong>for</strong> detection of Haemonchus contortus.<br />
Entomol 11:342-348. Helminthologia 33:73-75.<br />
Montenegro James S, Guillen A T and Toro M. 1992. Dot-ELISA<br />
para el diagnostico serologico de la anaplasmosis y<br />
babesiosis bovina. Rev. Ciesstifica FCV de Luz II ,23-29.<br />
Parija SC. 1998. A review of some simple immunoassays in the<br />
serodiagnosis of cystic hydatid disease. Acta Trop 70:17-24.<br />
Sriveny D, Raina OK, Yadav SC, Chandra D, Jayraw AK, Singh<br />
M, Velusamy R and Singh BP. 2006. Cathepsin L-cysteine<br />
proteinase in the diagnosis of bovine Fasciola gigantica<br />
infection. Vet Parasitol 135:25-31.<br />
Su XZ and Prestwood AK. 1991. A Dot-ELISA mimicry western<br />
blot test <strong>for</strong> the detection of swine trichinellosis. J Parasitol<br />
Prasad A, Nasir, A and Singh N. (2007). Dot-ELISA <strong>for</strong> the<br />
77:76-82.<br />
detection of preclinical Haemonchus contortus infection in<br />
sheep by using an adult somatic antigen and an Vercammen F, Berkvens D, Brandt J and Vansteenkiste W. 1998.<br />
immunoaffinity purified fraction. J Para Dis 31:22-28.<br />
A sensitive and specific 30-min Dot-ELISA <strong>for</strong> the detection<br />
of anti-leishmania antibodies in the dog. Vet Parasitol 79:221-<br />
Prasad A, Nasir A and Singh N.<strong>2008</strong>. Detection of anti-<br />
228.<br />
Haemonchus contortus antibodies in sheep by Dot-ELISA<br />
with immunoaffinity purified fraction of ES antigen during Wanduragala L, Kakoma I, Clabaugh GW, Abeygunawardena I,<br />
prepatency. Ind J Exp Biol 46:94-99.<br />
Levy MG, Ristic M. 1987. Development of Dot-Enzyme<br />
immunoassay <strong>for</strong> diagnosis of canine babesiosis. Am J Trop<br />
Reynaldo Dietze, Aloisio Falqueto, Luiz Carlos Pedrosa Valli,<br />
Med Hyg 36:20-21.<br />
Temizio Pereira Rodriques, Marcos Boulos and Ralph Corey.<br />
1995. Diagnosis of Canine Visceral Leishmaniasis with a Zimmerman GL, Nelson MJ and Clark CRB. 1985. Diagnosis of<br />
Dot-Enzyme-Linked Immunosorbent Assay. Am J Trop Med ovine fasciolosis by a dot ELISA: A rapid diagnostic<br />
Hyg 53:40-42. technique Am J Vet Res 46:1513-1515.<br />
Sood ML, Kaur G, Parmar A and Kapur J. 1996. Development of
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 15-21<br />
Original paper<br />
J P D<br />
Lymphatic filariasis in Benue State, Nigeria: community<br />
diagnosis using the rapid-<strong>for</strong>mat antigen immunochromatographic<br />
card test<br />
1 2<br />
E. A. Omudu and F. C. Oka<strong>for</strong><br />
1<br />
Department of Biological Sciences, Benue State University, Makurdi, Nigeria.<br />
2<br />
Department of Zoology, University of Nigeria, Nsukka, Nigeria.<br />
Received 16 July 2007; accepted 10 January <strong>2008</strong><br />
ABSTRACT. <strong>The</strong> whole blood immunochromatographic card test (ICT) <strong>for</strong> bancroftian filariasis<br />
was used in a survey conducted in Benue State, Nigeria. Eight hundred and <strong>for</strong>ty five persons in<br />
rural communities were examined, and 264 (31.2 %) of them tested positive <strong>for</strong> circulating filarial<br />
antigen (CFA). <strong>The</strong> persons in the age bracket of 40–49 years recorded the highest infection rate; it<br />
2<br />
was significantly higher in males (X = 7.02, df = 1, p < 0.05). Characteristics of the ICT card test<br />
procedure including high sensitivity, promptness and simplicity in field application justify its use<br />
<strong>for</strong> screening of filariasis. However, the test kits are expensive, and thus surveys based on this<br />
procedure are relatively limited. Further studies designed to use this technique on a wider scale in<br />
different bio-geographical areas in Nigeria are necessary to generate data regarding the<br />
distribution and prevalence of the disease.<br />
Keywords: community diagnosis ICT card test, lymphatic filariasis, Nigeria.<br />
INTRODUCTION<br />
22.1% of its population is thought to be infected<br />
(Michael et al., 1996). Although, mortality from<br />
lymphatic filariasis is low, the disease is the fourth<br />
leading cause of permanent disability and among the<br />
tropical diseases, lies second only to malaria in terms<br />
of disability-adjusted life years (WHO, 2002).<br />
Unprecedented and unplanned urbanization, over-<br />
crowding and deteriorating sanitary conditions in both<br />
urban and rural areas is believed to be exacerbating the<br />
transmission of the disease in Nigeria (Eigege et al.,<br />
2002; Braide et al., 2003; Terranella et al., 2006;<br />
Nwoke et al., 2006).<br />
Lymphatic filariasis, caused by Wuchereria bancrofti<br />
infection, is estimated to infect at least 128 million<br />
persons worldwide. It is a major cause of acute as well<br />
as chronic morbidity and incapacitation among the<br />
poorest 20% of the world's poor in tropical and subtropical<br />
zones of the world (Michael and Bundy,<br />
1997). Lymphatic filariasis is the second leading cause<br />
of permanent and long-term disability in the world,<br />
inflicting serious public health and socio-economic<br />
problem on the people (Terranella et al., 2006).<br />
Nigeria is the third most lymphatic filariasis endemic<br />
country in the world after India and Indonesia, and<br />
Corresponding author: Dr. Edward Agbo Omudu, Department of<br />
Biological Sciences, Benue State University, P M B 102119 Makurdi,<br />
Nigeria. E-mail: eddieomudu@yahoo.com<br />
<strong>The</strong> global ef<strong>for</strong>ts to eliminate lymphatic filariasis as a<br />
public health problem focus on mass drug<br />
administration (MDA), which is currently<br />
recommended in communities known or suspected to<br />
have a prevalence of lymphatic filariasis greater than
16<br />
Omudu and Oka<strong>for</strong><br />
1% ( Eigege et al., 2002). To determine community blood <strong>for</strong> the circulating Og4C3 antigen of W.<br />
eligibility <strong>for</strong> MDA, community mapping must be bancrofti has raised hope <strong>for</strong> quick community<br />
conducted either through parasitological or diagnosis (Weil et al., 1997). <strong>The</strong> ICT test is very<br />
serological investigation. <strong>The</strong> parasitological sensitive and specific to lymphatic filariasis that<br />
investigation is extremely difficult because it requires detects the infection within minutes, at any time of the<br />
collection of night blood samples; this is particularly day, without the need <strong>for</strong> laboratory facilities. Several<br />
problematic in many endemic communities in Africa comparative studies have been conducted to compare<br />
with strong religious and superstitious beliefs ICT test with other existing serological diagnostic<br />
(Gyapong et al., 1996; Haddix and Kestler 2000; options (Wamae et al., 1995; Weil et al., 1997;<br />
Braide et al., 2003). Antigen testing, there<strong>for</strong>e, Simonsen and Dunyo, 1999; Bhumiratana et al., 2000;<br />
became the recognized method of choice <strong>for</strong> the Eigege et al., 2002). <strong>The</strong> ICT has been found to be the<br />
detection of W. bancrofti infection. Unlike tests that most appropriate test <strong>for</strong> the field studies in Africa, and<br />
detect microfilariae, antigen tests can be per<strong>for</strong>med has been adopted as the method of choice <strong>for</strong><br />
with blood collected during the day or night. However, community mapping to facilitate implementation of<br />
existing enzyme-linked immunosorbent assay new strategies by the World Health Organization <strong>for</strong><br />
(ELISA) test <strong>for</strong> filarial antigenemia is difficult to the control and elimination of lymphatic filariasis. We<br />
per<strong>for</strong>m in the field, and has resulted in its limited use undertook this study to determine the prevalence of<br />
in endemic countries.<br />
the disease in Benue State, where there is a dearth of<br />
hospital and epidemiological records on the extent and<br />
<strong>The</strong> development of a rapid immunochromatographic<br />
distribution of the disease.<br />
card test (ICT) that qualitatively tests finger prick<br />
8 o 00 /<br />
8 o 30/<br />
9 00 /<br />
NIGERIA<br />
N A S A R A W A S T A T E<br />
o<br />
8 00<br />
/<br />
A G A T U<br />
G U M A<br />
Benue<br />
State<br />
TARABA<br />
K O G I<br />
o /<br />
7 30<br />
S T A T E<br />
G W E R<br />
MAKURDI<br />
A P A<br />
W E S T<br />
G W E R<br />
O T U K P O E A S T<br />
TARKA L O G O<br />
BURUKU<br />
G B O K O<br />
U K U M<br />
STATE<br />
o /<br />
7 30<br />
O H I M I N I<br />
K A T S I N A - A L A<br />
o /<br />
7 00<br />
U S H O N G O<br />
VANDE-<br />
IKYA K W A N D E<br />
OGBA-<br />
DIBO<br />
ENUGU<br />
OKPOKWU<br />
A D O<br />
O B I<br />
O J U<br />
KONSHISHA<br />
o 9 30 /<br />
N<br />
o /<br />
7 00<br />
STATE<br />
STATE BOUNDARY<br />
L.G. BOUNDARY<br />
WATER WAYS<br />
EBONYI<br />
STATE<br />
8 o 00 /<br />
o /<br />
8 30<br />
NATIONAL BOUNDARY<br />
SELECTED LOCAL<br />
GOVERNMENT AREAS<br />
LGAs NOT COVERED IN THE<br />
R.A.PROCEDURES<br />
CROSS RIVER STATE<br />
o /<br />
9 00<br />
Fig. 1. Local Government Areas where the ICT screening was conducted.<br />
CAMEROON<br />
REPUBLIC<br />
Kilometre<br />
0 15 30 40 45<br />
o<br />
6 o 30 /
Diagnosis of lymphatic filariasis<br />
17<br />
MATERIALS AND METHODS<br />
Description of the study area: Benue State is one of<br />
the 36 states in the Federal republic of Nigeria, a<br />
tropical country on the west coast of Africa. <strong>The</strong> state<br />
derives its name from River Benue, the second largest<br />
river in the country, and is located in the central region<br />
of the country where it lies between latitude 7° 13 and<br />
7° 49 and longitude 8° 15 and 8° 42 (Fig. 1). <strong>The</strong> State<br />
covers an area of about 34,059 square km with a<br />
population of over 4.2 million (NPC 2006). Majority<br />
of the inhabitants live in rural agricultural areas and<br />
engage in peasant agriculture, the State's reputation as<br />
the food-basket of the nation is being seriously<br />
jeopardized by the socio-economic consequences of<br />
parasitic diseases.<br />
<strong>The</strong> study was conducted in 6 out of 23 Local<br />
Government Areas (LGAs) of the State. Permission to<br />
conduct this study was granted by the state Ministry of<br />
Health and Human Services in Makurdi, the<br />
administrative headquarter of the State. In<strong>for</strong>med<br />
consent was obtained from all the participants after the<br />
explanation of the procedures and the likely benefits<br />
of the study. After explaining the purpose of the study<br />
to the village-chief and the traditional-leadership<br />
council and obtaining their permission and consent, all<br />
the participating adults (16 years of age and older)<br />
were asked to gather at the village Primary Health<br />
Care (PHC) Centre and randomly selected. <strong>The</strong><br />
individual verbal consent of the patients was obtained,<br />
following which they were assigned identification<br />
numbers and their names, age, occupation and marital<br />
status were recorded. <strong>The</strong>n after the collection of<br />
demographic data, ICT was per<strong>for</strong>med as per the<br />
manufacturer's instructions (NOW, ICT filariasis kits,<br />
Binax, Portland, ME, USA). <strong>The</strong> patient's left index<br />
finger was cleaned with methylated-spirit, and then<br />
punctured by using a sterile lancet. <strong>The</strong> initial sample<br />
of blood was removed using a cotton swab, and<br />
sufficient fresh blood was then collected to fill a 100-<br />
50<br />
45<br />
percentage positive<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
Series1<br />
10-19<br />
20-29<br />
30-39<br />
40-49<br />
50-59<br />
60-69<br />
70-above<br />
age-specific results of the ICT card test<br />
in study area<br />
Fig. 1. Age-specific results of ICT in study area.
18<br />
Omudu and Oka<strong>for</strong><br />
ql capillary tube. <strong>The</strong> blood was then transferred from ecological conditions and anthropogenic factors that<br />
the capillary tube to the pad on the ICT card and then may act as risk factors in the areas studied. Drainage<br />
sealed. <strong>The</strong> results of each ICT were read after 15 min. system and farming practice was similar and 66.2% of<br />
Result was considered positive, when two pink lines the participants were farmers.<br />
appeared on the card's window and negative when<br />
only a single line was seen. Test results with the<br />
DISCUSSION<br />
individual's identification code were recorded on the <strong>The</strong> assessment of filariasis burden in a community is<br />
patients' diagnostic data sheet.<br />
a pre-requisite <strong>for</strong> planning, implementing and<br />
Data analysis: Data were entered into a computer and<br />
evaluation of intervention strategies. Sensitivity,<br />
statistical analysis was carried out in Epi Info, version<br />
specificity, speed and simplicity are the important<br />
2002 (Centre <strong>for</strong> Disease Control and Prevention,<br />
factors considered in assuring efficiency of screening<br />
Atlanta). Chi-Square tests were used to compare and<br />
<strong>for</strong> bancroftian filariasis. <strong>The</strong> assessment of field<br />
test <strong>for</strong> significance.<br />
per<strong>for</strong>mance and field applicability of new diagnostic<br />
methods in Africa is an important step towards<br />
RESULTS<br />
stopping transmission. Our findings are similar to<br />
those of others reported in Africa ( Ramzy et al., 1999;<br />
Of the 845 participants, 264 (31.2%) tested positive<br />
Eigege et al., 2002; Terranella et al., 2006) and<br />
<strong>for</strong> circulating filarial antigen (CFA). <strong>The</strong> sex of the<br />
elsewhere in Brazil, Sri Lanka and India (Pani et al.,<br />
participant appeared to have a bearing on their<br />
2000; Braga et al., 2003; Weerasooriya et al., 2003).<br />
infection as the males had significantly higher<br />
<strong>The</strong> overall prevalence of filarial antigenaemia was<br />
infection rate (36.3 %) than the female participants<br />
very high (31.2%), and this is much higher than earlier<br />
2<br />
(25.4%; X = 7.43, df = 1, p < 0.05). Ado LGA recorded reported and far exceeds the World Health<br />
the highest prevalence with 53.7% of sampled Organization's threshold of 1% positivity, <strong>for</strong> MDA in<br />
population testing positive, whereas Ogbadibo a given community ( WHO, 1998). <strong>The</strong> high<br />
recorded only 2.6% prevalence (Table I); this endemicity of the disease in Benue State may be as a<br />
2<br />
difference was statistically significant ( X = 9.81, df = result of the deteriorating sanitary conditions, which<br />
6, p < 0.05). <strong>The</strong> highest age-related prevalence was create breeding sites <strong>for</strong> mosquitoes, the slow and<br />
recorded in the age group of 40–49 years (Table II); ineffective pace of the distribution and availability of<br />
however, there was no statistically significant Insecticide Treated Bednets (ITBNs) in the Roll Back<br />
difference in the prevalence of antigenaemia Malaria programme and difficulties in diagnosis and<br />
according to age group. Of the 21 villages where the treatment of the disease in many rural health centers.<br />
study was conducted, ICT-detected prevalence ranged<br />
from 0–57.1%. Three villages (Rijo, Ofuke and <strong>The</strong> bearing of gender in ICT positivity and by<br />
Odiapa in Ado, Apa and Obi LGAs, respectively) had a implication the prevalence of lymphatic filariasis<br />
filarial antigenaemia prevalence rate of > 30%, and observed in this study, is in consonance with other<br />
93.3% of participants were clinically asymptomatic findings in North Central Nigeria and Brazil (Eigege<br />
(showing no visible signs of lymphatic filariasis). et al., 2002; Braga et al., 2006). <strong>The</strong> reasons <strong>for</strong> this<br />
Nevertheless, we encountered 6 participants who had gender bearing should generate a lot of research<br />
elephantiasis but ICT gave negative results.<br />
interest because both males and females are equally<br />
exposed to mosquito bites and other risk factors in a<br />
Investigation of treatment history revealed that 68.2% given community. <strong>The</strong> higher ICT positive results<br />
of the total sample population had taken ivermectin <strong>for</strong> obtained within the age bracket of 40–49 also agrees<br />
the treatment of onchocerciasis, and 51.1% of those with age-related findings from Nigeria and elsewhere<br />
who had taken ivermectin had been on the treatment (Udonsi, 1986; Meyrowitsch et al., 1995; Surendran<br />
<strong>for</strong> at least 2 years. Though ICT was easy to use in the et al., 1996; Eigege et al., 2002). <strong>The</strong> age-related<br />
field and was well-accepted by participating prevalence of lymphatic filariasis in endemic areas<br />
communities; however, we observed 13 cases whose appears to be low among subjects less than 40 years in<br />
card exhibited only one pink line after 15 min in the age and higher in individuals above 40. This is due to<br />
field but a slight pink second line appeared during increased contact between older subjects and the<br />
post-field analysis. <strong>The</strong>se cases were recorded as mosquito vectors, as a result of long exposure during<br />
negative in accordance with the manufacturer's outdoor activities such as farming and fishing.<br />
instructions. <strong>The</strong>re is no marked difference in
Diagnosis of lymphatic filariasis<br />
19<br />
Table I. Prevalence of lymphatic filariasis based on ICT according to sex<br />
Male Female Total<br />
LCA Number Number Percentage Number Number Percentage Number Number Percentage<br />
examined positive (%) examined positive (%) examined positive (%)<br />
Ado 81 44 61.7 66 32 48.5 147 76 55.8<br />
Apa 80 30 37.5 51 9 17.6 131 39 29.8<br />
Gwer 72 29 40.3 29 11 37.9 101 40 39.6<br />
West<br />
Obi 41 22 53.6 69 26 37.7 110 48 43.6<br />
Ogbadibo 52 2 3.84 69 1 1.45 121 3 2.47<br />
Okpokwu 52 10 19.2 43 7 16.3 95 17 17.9<br />
Ushongo 75 19 25.3 76 22 28.9 151 41 27.1<br />
Total 453 156 35.7 403 108 26.8 856 264 31.2
20<br />
Omudu and Oka<strong>for</strong><br />
<strong>The</strong> per<strong>for</strong>mance of ICT has been found to be need <strong>for</strong> complicated laboratory procedures and no<br />
comparable to that of the conventional finger-prick, need <strong>for</strong> specialized technicians. Its usage in other<br />
night-blood thick smear examination ( Pani et al., parts of Nigeria will help generate the much needed<br />
2000; Braga et al., 2003). Among its several epidemiological in<strong>for</strong>mation on the distribution and<br />
advantages over the conventional technique are that it prevalence of lymphatic filariasis in the country.<br />
can be per<strong>for</strong>med on the whole-blood sample<br />
collected by finger-prick at any time of the day and the<br />
Conflict of interest: <strong>The</strong> authors declare that they<br />
results are instant. <strong>The</strong> ICT can also be stored after use<br />
have no conflict of interest<br />
and then later used to evaluate treatment progression. Authors' contribution: Dr. Omudu Edward and<br />
<strong>The</strong> ICT has also per<strong>for</strong>med considerably better than Prof. Oka<strong>for</strong> Fabian both designed the study. <strong>The</strong> later<br />
many other immunological techniques including conducted the field study, analysis and preparation of<br />
immunoradiometric assay and enzyme linked the manuscript under the supervisionof the <strong>for</strong>mer.<br />
immunosorbent assay (ELISA; Weil et al., 1997;<br />
Simonsen and Dunyo, 1999; Ramzy et al., 1999; Acknowledgement: We would like to thank Alves<br />
Weerasooriya et al., 2003). Because the detection of Nina of Binax Incorporate Scarborough, ME, USA,<br />
CFA is a useful non-microscopic method <strong>for</strong> the manufacturers of ICT filariasis test-kits <strong>for</strong><br />
diagnosis of bancroftian filariasis that is currently facilitating the purchase of the test kits and helping<br />
making the transition from the research laboratory to with the logistics of sending them to Nigeria.<br />
the field, the ICT surpasses these other immunological<br />
techniques in field application and simplicity.<br />
REFERENCES<br />
Gyapong et al. (1998) and Eigege et al. (2002) have all Bhumiratana A, Koyadun S, Rojanapremsuk J and<br />
Suvannadabba S. 2000. Diagnosis of Wuchereria bancrofti<br />
reported positive correlation between ICT results and<br />
infection in endemic populations: diagnostic approaches to<br />
community hydrocoele rates in Africa.<br />
control and elimination. Mandihol J 7:101-107.<br />
Notwithstanding the brilliant per<strong>for</strong>mance of ICT, its Braga C, Dourado MI, Ximenes RA, Alves L, Brayner F, Rocha<br />
large-scale field use in control programmes in A and Alexander N. 2003. Field evaluation of the whole<br />
endemic countries (especially in poor African and blood immunochromatographic test <strong>for</strong> rapid bancroftian<br />
Southeast Asian countries) will require a<br />
filariasis diagnosis in the Northeast Brazil. Rev Inst Med<br />
Trop S Paulo 45: 125-129.<br />
downward review of its cost. As at the end of<br />
2006, only very few countries in Africa have been able Braide EI, Ikpeme B, Edet E, Atting I and Kale OO. 2003.<br />
to conduct their lymphatic filariasis mapping exercise, Preliminary observations on the occurrence of lymphatic<br />
filariasis in Cross Rivers state, Nigeria. Nigeria J Parasitol<br />
a crucial component of national eradication<br />
24:9-16.<br />
programmes. <strong>The</strong> slow pace of mapping filariasis is as<br />
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,<br />
village in Nigeria is estimated to cost well over US $ Umaru J, Jinadu MY, Matthias W, Amadiegwu S and<br />
Hopkins DR. 2002. Rapid Assessment <strong>for</strong> lymphatic<br />
100,000 (Eigege et al., 2002). In India, the cost of an<br />
filariasis in central Nigeria: a comparison of the<br />
ICT kit alone is between US $ 1.05 and US $ 1.29, it is<br />
immunochromatographic card test and hydrocoele rates in<br />
estimated that 500 tests will cost US $ 120 (Pani et al., an area of high endemicity. Am J Trop Med Hyg 68:643-646.<br />
2000). In Sri Lanka, cost analysis of the kit showed<br />
Gyapong M, Gyapong JO, Adjei S, Vlassoff C and Weiss M.<br />
that ICT costs US $ 2.75 per unit compared to thick-<br />
1996. Filariasis in Northern Ghana: some cultural beliefs<br />
blood film, which is US $ 0.30 (Braga et al., 2003). and practices and their implications <strong>for</strong> disease control. Soc<br />
<strong>The</strong> World Health Organization may consider Sci Med 43:235-242.<br />
subsidizing the cost of ICT kits and facilitating<br />
Gyapong JO, Webber RH, Morris J and Bennetts S. 1998.<br />
improved access to the technology <strong>for</strong> poor countries,<br />
Prevalence of hydrocoele as a rapid diagnostic index <strong>for</strong><br />
where the disease is most endemic. <strong>The</strong> other area of lymphatic filariasis. Trans R Soc Trop Med Hyg 98:40-43.<br />
challenge is the storage requirement, as the ICT cards<br />
need to be stored at 2°C to 8°C, and this may be Haddix A and Kestler A. 2000. Lymphatic filariasis: economic<br />
aspects of the disease and programme <strong>for</strong> its elimination.<br />
difficult in many rural areas in the tropics.<br />
Trans R Soc Trop M Hyg 94:592-593.<br />
Despite these challenges, the features of the ICT<br />
proved to be extremely advantageous: its high<br />
sensitivity, the ability to offer prompt diagnosis, no<br />
Nwoke BEB, Dozie INS, Jiya J, Saka Y, Ogidi JA, Istifanus WA,<br />
Mafiana CF, Oyene U, Amali O, Ogbu-Pearce P and Nuttal I.<br />
2006. <strong>The</strong> prevalence of hydrocoele in Nigeria and its
Diagnosis of lymphatic filariasis<br />
21<br />
implication on mapping of lymphatic filariasis. Nigerian J<br />
Parasitol 27:29-35.<br />
Michael E, Bundy DAP and Grenfell BT. 1996. Re-assessing<br />
the global prevalence and distribution of lymphatic<br />
filariasis. Bull WHO 75:491-503.<br />
Michael E and Bundy DAP. 1997. Global mapping of<br />
lymphatic filariasis. Parasitol Today 13:471-476.<br />
Ramzy RMR, Helmy H, El-Lethy AST, Kandil AM, Ahmed ES,<br />
Weil GJ and Faris R. 1999. Field evaluation of a rapid<strong>for</strong>matkit<br />
<strong>for</strong> the diagnosis of bancroftian filariasis in Egypt.<br />
East Mediterr Health J 5:880-887.<br />
Terranella A, Eigege A, Gontor P, Dagwa P, Damishi S,<br />
Blackburn B, McFarland, D. Zingeser, J. Jinadu, M.Y., Miri,<br />
E. and Richards F.O. (2006). Urban lymphatic filariasis in<br />
central Nigeria. Ann Trop Med Parasitol 100:1-10.<br />
Udonsi JK. 1986. <strong>The</strong> status of human filariasis in relation to<br />
clinical signs in endemic areas of the Niger Delta. Ann Trop<br />
Med Parasitol 80:425-432.<br />
Weil GJ, Lammie PJ and Weiss N. 1997. <strong>The</strong> ICT filariasis test:<br />
a rapid–<strong>for</strong>mat antigen test <strong>for</strong> the diagnosis of Bancroftian<br />
filariasis. Parasitol Today 13:401-404.<br />
Weerasooriya MV, Itoh M, Kimura E and Fujimaki Y. 2003.<br />
Human infection withWuchereria bancrofti in Matara, Sri<br />
Lanka: <strong>The</strong> use of ELISA to detest 1g G4<br />
in urine and ICT and<br />
test to detect antigen in blood. Ann Trop Med Parasitol<br />
97:179-185.<br />
Pani SP, Hoti SL, Elango A, Yuvaraj J, Lall R and Ramaiah KD.<br />
2000. Evaluation of the ICT whole blood antigen card test to<br />
detect infection due to nocturnally periodic Wuchereria<br />
bancrofti in south India. Trop Med Int Health 5:359-363.<br />
World Health Organization. 1998. Report of a WHO In<strong>for</strong>mal<br />
Consultation on Epidemiologic Approaches to Lymphatic<br />
Filariasis Elimination: Initial Assessment, Monitoring and<br />
Certification. Geneva: WHO.<br />
Meyrowitsch DW, Simonsen PE and Makunde WH. 1995.<br />
Bancroftian filariasis: analysis of infection and disease in<br />
five endemic communities in north-eastern Tanzania. Ann<br />
Trop Med Parasitol 89:653-663.<br />
Surendran K, Pani SP, Soudarssanane MB, Spinivasa DK,<br />
Bordolai AI and Subramania S. 1996. Natural history, trend<br />
of prevalence and spectrum of manifestations of bancroftian<br />
filarial disease in Pondicherry (South India). Acta Tropica<br />
61:9-18.<br />
Simonsen PE and Dunyo SK. 1999. Comparative evaluation of<br />
three new tools <strong>for</strong> diagnosis of bancroftian filariasis based<br />
on detection of specific circulating antigens. Trans R Soc<br />
Trop Med Hyg 93:278-282.<br />
Wamae CN, Lammie PJ and Muttunga JN. 1995. Bancroftian<br />
filariasis: profile of serum antifilarial antibody and<br />
circulating parasite antigen. East Afr Med J 72:492-494.
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 22-29<br />
Original paper<br />
J P D<br />
Purification and characterization of Plasmodium berghei<br />
glutathione reductase<br />
1 2 1<br />
G. Kapoor , U. Bagai and H. S. Banyal<br />
1<br />
Department of Biosciences, Himachal Pradesh University, Shimla.<br />
2<br />
Department of Zoology, Panjab University, Chandigarh.<br />
Received 18 January <strong>2008</strong>; accepted 31 March <strong>2008</strong><br />
ABSTRACT. Glutathione (GSH) is a major low molecular mass thiol present in many organisms<br />
and helps to maintain a reduced intracellular environment. GSH and glutathione reductase (GR)<br />
act as an antioxidant defense system by protecting the cell from the damage caused by free-radicals.<br />
+<br />
Glutathione reductase (glutathione: NADP oxidoreductase E.C. 1.8.1.7; GR) was analysed in<br />
rodent malaria parasite, Plasmodium berghei and purified through ammonium sulphate<br />
fractionation and Sephadex G-200. Cell-free P. berghei showed specific activity of 0.617±0.08<br />
units/mg protein. Maximum GR activity was observed in cytosolic fraction of P. berghei. <strong>The</strong><br />
molecular weight of parasite enzyme was estimated to be 25 kDa and it was maximally active at pH<br />
6.8 and at 25–40°C. Km and Vmax values <strong>for</strong> oxidised glutathione (GSSG) substrate were 0.02 mM<br />
and 0.5 mM, respectively. Methylene blue non-competitively inhibited the enzyme and its Ki was 0.4<br />
mM.<br />
Keywords: enzyme characterization, glutathione reductase, malaria parasite, Plasmodium berghei<br />
INTRODUCTION<br />
Plasmodium has been considered very sensitive to<br />
oxidative stress. During the erythrocytic stages of its<br />
life cycle, the parasite is subjected to an increased<br />
oxidative attack by reactive oxygen species (ROS)<br />
produced by the parasite, red cells and host immune<br />
response (Atamna and Ginsburg, 1993). To combat<br />
this stress the parasite relies upon antioxidant<br />
enzymes like glutathione reductase, thioredoxin<br />
reductase, superoxide dismutase, glutathione<br />
transferase etc. <strong>The</strong> tripeptide glutathione (GSH) is a<br />
major low molecular mass thiol in almost all<br />
eukaryotes with various cellular functions. GSH<br />
Corresponding author: Prof. H. S. Banyal, Department of<br />
Biosciences, Himachal Pradesh University, Shimla-171 005,<br />
India. Email: hsbanyal@yahoo.co.in<br />
regulates the metabolism of proteins and their<br />
activities through thiol-disulphide exchange, plays a<br />
role in antioxidant defense and in maintaining a<br />
reduced environment of the cytosol (Becker et al.,<br />
2003).<br />
Glutathione reductase (GR; E.C. 1.8.1.7), a member of<br />
the pyridine nucleotide disulphide oxidoreductase<br />
family of flavoenzyme catalyses the reduction of<br />
oxidised glutathione (GSSG) to reduced <strong>for</strong>m (GSH)<br />
in the presence of nicotinamide adenine di-nucleotide<br />
phosphate reduced (NADPH). Reduced glutathione is<br />
used in the detoxification of xenobiotics, protecting<br />
thiol groups of intracellular proteins, scavenging<br />
hydrogen peroxide and other organic peroxides, and<br />
helps in maintaining a reduced environment inside the<br />
cytosol. GR has been studied in human erythrocytes<br />
(Worthington and Rosemeyer, 1974; Le Trang et al.,
Plasmodium berghei GR<br />
23<br />
1983), bovine erythrocytes (Erat et al., 2003), sheep determined spectrophotometrically in a double beam<br />
liver (Ulusu et al., 2005) and Plasmodium falciparum spectrophotometer (Perkin Elmer) at room<br />
(Krauth-Siegal et al., 1996). In the present study GR of temperature according to the modified method of<br />
P. berghei has been purified and some of its Worthington and Rosemeyer (1974). <strong>The</strong> 2 ml assay<br />
characteristics have been investigated.<br />
system contained 0.01M potassium phosphate buffer,<br />
MATERIALS AND METHODS<br />
pH 7.2, 0.2 mM EDTA, 0.1 mM NADPH and an<br />
appropriate volume of enzyme extract, and the<br />
Parasite: P. berghei (NK 65) was maintained in reaction was initiated by the addition of 0.1 mM GSSG<br />
BALB/c mice, and the course of parasitaemia was at room temperature. <strong>The</strong> oxidation of NADPH<br />
monitored in Giemsa-stained thin blood smears. resulting in decrease in absorbance was monitored at<br />
Normal or P. berghei-infected (about 50% 340 nm. One enzyme unit is defined as the oxidation of<br />
parasitaemia) mice were anaesthetized with diethyl 1µ mol NADPH/min under the assay conditions.<br />
ether and blood was collected in citrate saline.<br />
Protein was determined according to Lowry et al.<br />
(1951), and specific activity of GR was units of<br />
Enzyme extract: <strong>The</strong> citrated-blood from normal or enzyme/mg protein.<br />
P. berghei-infected mice was centrifuged at 1,000 x g<br />
o<br />
<strong>for</strong> 10 min at 4 C. <strong>The</strong> plasma was then aspirated, and Ammonium sulphate fractionation: <strong>The</strong> cell-free<br />
the pellet suspended in equal volume of phosphatewith<br />
ammonium sulphate in steps of 0%-20%, 20%-<br />
parasite homogenate was subjected to precipitation<br />
buffered saline (PBS, 0.01M), pH 7.2, and loaded onto<br />
a CF-11 cellulose (Whatmann) column (1.5 cm x 20 40%, 40%-80% and 80%-100% saturation. <strong>The</strong><br />
cm) to remove leukocytes (Sharma and Banyal, 2007). precipitates of each cut were dissolved in a minimum<br />
<strong>The</strong> eluted leukocyte-free blood was centrifuged at volume of 50 mM potassium phosphate buffer pH 7.2<br />
1,000 x g <strong>for</strong> 10 min at 4°C. A part of the pellet was and dialyzed at 4ºC using the same buffer containing<br />
used <strong>for</strong> total erythrocytes, and the remaining pellet 1mM EDTA with 2–3 changes of buffer.<br />
was suspended in an equal volume of saponin in PBS Gel filtration on Sephadex G-200: Dried Sephadex<br />
o<br />
(0.2% w/v), pH 7.2 and incubated <strong>for</strong> 30 min at 4 C G-200 (Sigma) was swollen in distilled water <strong>for</strong> 72 h<br />
with intermittent mixing. <strong>The</strong> suspension was then at 4°C. A glass column was filled with Sephadex G-<br />
o<br />
centrifuged at 15,000 x g <strong>for</strong> 20 min at 4 C. <strong>The</strong> 200 (1.5 cm x 20 cm) and a flow rate of 12 ml/h was<br />
haemolysate thus obtained was aspirated, while maintained. <strong>The</strong> column was equilibrated with 50 mM<br />
erythrocyte membranes from normal mice were potassium phosphate buffer, pH 7.2 containing 1mM<br />
washed thrice with PBS. In case of infected animals, EDTA until the final absorbance difference became<br />
the erythrocyte membranes overlying cell-free zero at 280 nm. <strong>The</strong> dialyzed sample was loaded onto<br />
parasites were separated. Both the preparations were the column and elutions were collected in 1 ml volume<br />
washed thrice with PBS (Banyal and Fitch, 1982). <strong>The</strong> at 4°C. In each fraction enzyme activity and protein<br />
preparation from normal mice yielded total were determined.<br />
erythrocytes, haemolysate and erythrocyte<br />
membranes, and infected blood was fractionated in the Characterization of GR: Sodium dodecyl sulphate<br />
above three components and cell-free parasites also. polyacrylamide gel electrophoresis (SDS-PAGE).<br />
<strong>The</strong> total erythrocytes, haemolysate, erythrocyte Purified enzyme was electrophoretically<br />
membranes and cell-free parasite were suspended in characterized using sodium dodecyl sulphate<br />
an appropriate volume of 0.01M PBS, pH 7.2, polyacrylamide gel electrophoresis by the method of<br />
homogenized in Potter Elvehjem homogenizer at 4ºC Laemmli (1970) in a mini vertical slab gel apparatus<br />
o<br />
and centrifuged at 1,000 x g <strong>for</strong> 10 min at 4 C, and the (Genei, Bangalore) as given by Banyal and Inselburg<br />
supernatant was used as an enzyme extract.<br />
(1985) employing 3% stacking and 10% separating<br />
gels. <strong>The</strong> gels were stained in silver nitrate (Merril et<br />
Subcellular fractionation: Cell-free P. berghei was al., 1981). <strong>The</strong> pH <strong>for</strong> optimum activity of P. berghei<br />
homogenized at 4°C in pre-chilled 0.25 M sucrose GR was determined by using .01M potassium<br />
solution. <strong>The</strong> resulting homogenate was subjected to phosphate buffer <strong>for</strong> pH 5.5–8.0.<br />
differential centrifugation according to Banyal et al.<br />
(1979) and different fractions were obtained.<br />
To study the effect of temperature on GR activity,<br />
enzyme alone was first incubated <strong>for</strong> 10min at 0°C,<br />
Enzyme assay: Glutathione reductase activity was 25°C, 40°C, 80°C and 100°C (boiling water bath).
24 Kapoor et al.<br />
After incubation at desired temperature the sample was also noticed in other fractions.<br />
was transferred to ice bath so that further destruction<br />
of enzyme could be prevented. This treated enzyme<br />
P. berghei GR protein was mainly precipitated with<br />
was then assayed.<br />
40%-80% of ammonium sulphate resulting in 8.3 fold<br />
purification of the enzyme (Table III). When the<br />
Km and Vmax values <strong>for</strong> oxidised glutathione (GSSG) precipitated protein was dialyzed and loaded on to<br />
substrate was calculated from Lineweaver-Burk plot. Sephadex G-200 column the enzyme was eluted in<br />
Enzyme activity was assayed at different GSSG fractions 11 to 17 (Fig. 1) with a peak in fraction 13.<br />
concentrations with a fixed concentration of NADPH. This fraction contained 2.19 units/mg of enzyme.<br />
<strong>The</strong>re was a 14.5 fold purification of the enzyme by<br />
Ki <strong>for</strong> methylene blue was determined by incubating<br />
Sephadex G-200.<br />
purified enzyme with or without Methylene blue <strong>for</strong> 5<br />
min at room temperature and then assayed.<br />
SDS-PAGE analysis showed that molecular weight of<br />
RESULTS<br />
P. berghei GR was 25kDa (Fig.2). <strong>The</strong> enzyme was<br />
significantly active between pH 6.6 to 7.0 with<br />
P. berghei exhibited significant activity of glutathione optimum activity at 6.8 (Fig. 3). Preincubation of<br />
reductase (Table I). Parasite enzyme activity was enzyme at different temperatures showed that the<br />
about three fold higher compared to host erythrocytes. enzyme was active at lower temperatures while no<br />
Parasitized haemolysate contained more activity of activity observed when the protein treated at 100°C<br />
enzyme than the normal haemolysate. <strong>The</strong> host (Table IV). <strong>The</strong> Vmax and Km <strong>for</strong> GSSG were 0.5mM<br />
erythrocyte membranes were without any enzyme and 0.02mM respectively (Fig. 4). Methylene blue<br />
activity.<br />
non-competitively inhibited GR-GSSG enzyme<br />
substrate reaction with Ki of 0.4mM (Fig. 5).<br />
<strong>The</strong> enzyme was mainly confined to the cytosolic part<br />
of the parasite (Table II) though a very little activity DISCUSSION<br />
Table I. Activity of glutathione reductase in normal and P. berghei-infected erythrocytes and their fractions<br />
Erythrocyte fraction Normal P. berghei-infected<br />
(U/mg)<br />
(U/mg)<br />
Total 01781+ _ 0.02 0.186 + _ 0.02<br />
Haemolysate 0.170 + _ 0.02 0.342 +_ 0.40<br />
Erythrocyte membranes Nil Nil<br />
Cell-free parasite - 0.617 +_ 0.08<br />
Results are mean + _ SD of three experiments.<br />
Table II. GR activity in subcellular fractions of P. berghei<br />
Sediment<br />
Specific activity<br />
(U/mg)<br />
None (homogenate) 0789 + _ 0.11<br />
600xg 0.160 + _ 0.02<br />
10,000xg 0.370 +_ 0.19<br />
24,000xg 0.02 + 0.03<br />
105,000xg<br />
Supernatant 3.7 +_ 0.53<br />
Results are mean +_ SD of three experiments.<br />
Nil
Plasmodium berghei GR<br />
25<br />
Tabel III. Purification of P. berghei GR by ammonium sulphate and Sephadex G-200<br />
Fraction Volume Activity Protein Specific Purification<br />
(ml) (U) (mg/ml) activity (X/fold))<br />
(U/mg)<br />
Crude 14 1.07 1.06 0.3369 1.0<br />
0%-20% 2 Nil 10.0 Nil Nil<br />
20%-40% 2.5 Nil 5.7 Nil Nil<br />
40%-80% 3 8.6 3.0 2.8 8.3<br />
80%-100% 1.5 Nil 0.500 Nil Nil<br />
Sephadex<br />
eluate<br />
11 1 0.064 0.387 0.165 0.4<br />
12 1 0.321 0.354 0.906 2.6<br />
13 1 0.707 0.322 2.19 6.5<br />
14 1 0.385 0.516 0.746 2.2<br />
15 1 0.257 0.580 0.433 1.2<br />
16 1 0.192 0.419 0.458 1.3<br />
17 1 0.064 0.480 0.132 0.3<br />
Table IV. Effect of temperature on activity of P. berghei GR<br />
Temperature<br />
Specific activity (U/mg)<br />
-4°C 0.187<br />
0°C 0.187<br />
25°C 0.200<br />
40°C 0.200<br />
60°C 0.080<br />
80°C 0.026<br />
100°C 0.00<br />
<strong>The</strong> undesirable biological effects of oxidative agents oxidative stress since the parasite lacks catalase and<br />
like free radicals and reactive oxygen species are GSH peroxidases. Maximum activity of P. berghei GR<br />
eliminated by enzymatic and non-enzymatic was observed in cytosolic part of the parasite. GSH<br />
antioxidant defense systems. P. berghei parasitized dependent decomposition of toxic FP IX occurs in the<br />
host cells contained GR activity while the erythrocyte cytosol, hence maximum GR activity observed in<br />
membranes lacked this enzyme. Increased GR activity cytosolic fraction may be due to such requirement. A<br />
in P. berghei-infected erythrocytes indicates that GSH linked thioredoxin-glutathione system in the cytosolic<br />
is not taken up from the host but there is GSH synthesis and mitochondrial compartment has been<br />
due to the action of glutathione cycle in parasite. characterized in helminths also (Salinas et al., 2004).<br />
Krauth-Siegal et al (1996) also reported higher GR<br />
activity in P. falciparum-infected erythrocytes<br />
SDS-PAGE of purified GR revealed a subunit<br />
compared to normal host erythrocytes. Parasite's own<br />
molecular mass of 25kDa, however, P. falciparum GR<br />
GR activity enables the parasite to maintain its<br />
is reported to be a dimer protein of 105kDa (Krauth<br />
reducing environment efficiently under enhanced<br />
Siegal et al., 1982) with a subunit mass of 54.7kDa. h
26 Kapoor et al.<br />
2.5<br />
2.5<br />
Protein (mg/ml)<br />
2<br />
1.5<br />
1<br />
0.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
Specific activity (U/mg)<br />
0<br />
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<br />
Fraction number<br />
0<br />
Protein (mg/ml)<br />
Specific activity (U/mg)<br />
Fig. 1. Purification of glutathione reductase from cell-free P. berghei using Sephadex G-200.<br />
kDa<br />
97.4<br />
66<br />
43<br />
29<br />
Fig. 2.<br />
1 2 3 4<br />
SDS-PAGE of GR purified from P. berghei.<br />
Lane 1 = Protein standard<br />
Lane 2 = Cell-free parasite homogenate<br />
Lane 3 = Fraction 12<br />
Lane 4 = Fraction 13
Plasmodium berghei GR<br />
27<br />
0.2<br />
Specific activity (U/mg)<br />
0.16<br />
0.12<br />
0.08<br />
0.04<br />
0<br />
6.4 6.6 6.8 7 7.2 7.4 7.6 7.8<br />
pH<br />
Fig. 3. Effect of pH on activity of GR purified from P. berghei.<br />
Fig. 4. Effect of different concentrations of GSSG on activity of P. berghei GR.
28 Kapoor et al.<br />
Fig. 5. Ki determination of P. berghei GR using methylene blue.<br />
Taking into account the prosthetic group FAD P. Intraerythrocytic oxidative stress to parasite is<br />
falciparum GR was found to be a homodimer of combated with the help of antioxidant enzymes, which<br />
111kDa (Krauth Siegal et al., 1996) while human GR are essential <strong>for</strong> their survival. Disruption of<br />
is a dimer with a subunit mass of 52.3kDa (Becker et antioxidant enzymes like GR is a feasible way to<br />
al., 1991). <strong>The</strong> variation in enzyme molecular weight interfere with parasite development during<br />
of rodent and human malaria parasite may be due to erythrocytic schizogony and such enzymes may be<br />
different species or monomer and dimer nature of GR. exploited in <strong>for</strong>mulation of antimalarials to disrupt the<br />
P. berghei GR showed maximum activity in the propagation of malaria parasite.<br />
temperature range of 25ºC to 40ºC while at 80ºC GR<br />
activity was very little and the enzyme was maximally<br />
ACKNOWLEDGEMENTS<br />
active at pH 6.8. <strong>The</strong>se results are similar to GR from Mr. Gaurav Kapoor is grateful to ICMR, New Delhi<br />
P. falciparum and human red blood cell GR (Krauth <strong>for</strong> providing JRF-SRF.<br />
Siegal et al., 1996).<br />
REFERENCES<br />
Km of P. berghei GR <strong>for</strong> GSSG is 0.02mM and Vmax<br />
Atamna H and Ginsburg H. 1993. Origin of reactive oxygen<br />
0.5mM. Ulusu et al. (2005) reported Km value of<br />
species in erythrocytes infected with Plasmodium<br />
0.02mM <strong>for</strong> GSSG and 0.025mM <strong>for</strong> NADPH while falciparum. Mol Biochem Parasitol 61:231-241.<br />
Vmax was 0.255mM and 0.266mM <strong>for</strong> GSSG and<br />
Banyal HS and Fitch CD. 1982. Ferriprotoporphyrin IX<br />
NADPH respectively in sheep liver GR. Schistosoma<br />
binding substances and the mode of action of chloroquine<br />
digitata GR has been found to have Km value of against malaria. Life Sciences 31:1137-1142.<br />
27.4µM <strong>for</strong> GSSG (Muller et al., 1995). Methylene<br />
blue is a specific inhibitor of GR. Methylene blue nonof<br />
parasite inhibitory Plasmodium falciparum monoclonal<br />
Banyal HS and Inselburg J. 1985. Isolation and characterization<br />
competitively inhibited P. berghei GR with Ki of<br />
antibodies. Am J Trop Med Hyg 34:1055-1064.<br />
0.4mM.
Plasmodium berghei GR<br />
29<br />
Banyal HS, Pandey VC and Dutta GP. 1979. Subcellular Le Trang N, Bhargava KK, Cerami A. 1983. Purification of<br />
fractionation and localization of marker enzymes of glutathione reductase from gerbil liver in two steps. Anal<br />
erythrocytic stages of Plasmodium knowlesi. <strong>Indian</strong> J Biochem 133:94-99.<br />
Parasitol 3:9-14.<br />
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. 1951.<br />
Becker K, Kanzok SM, Lozef R, Fischer M, Schirmer RH and Protein measurement with Folin phenol regent. J Biol Chem<br />
Rahlfs S. 2003. Plasmoredoxin, a novel redox-active 193:265-275.<br />
protein unique <strong>for</strong> malarial parasites. Eur J Biochem<br />
270:1057-1064.<br />
Merril CR, Goldman D, Sedman SA and Ebert HM. 1981.<br />
Ultrasensitive stain <strong>for</strong> protein in polyacrylamide gel shows<br />
Becker K, Krebs B and Schirmer RH. 1991. Protein-chemical regional variation in cerebrospinal fluid proteins. Science<br />
standardization of the erythrocyte glutathione reductase 211:1437-1438.<br />
activation test. Application to hyperthyroidism. Int J Vit<br />
Nutr Res 61:180-187.<br />
Muller S, Walter RD and Fairlamb AH. 1995. Differential<br />
susceptibility of filarial and human erythrocyte glutathione<br />
Erat M, Sakiroglu H and Ciftci M. 2003. Purification and reductase to inhibition by trivalent organic arsenical<br />
characterization of glutathione reductase from bovine melarson oxide. Mol Biochem Parasitol 71:211-219.<br />
erythrocytes. Prep Biochem Biotechnol 33: 283-300.<br />
Salinas G, Selkirk ME, Chalar C, Maizels RM and Fernandez C.<br />
Krauth-Siegal RL, Blatterspiel R, Saleh M, Schulz GE, 2004. Linked thioredoxin-glutathione systems in<br />
Schirmer RH and Untuchi-Grau R. 1982. Glutathione platyhelminths. Trends Parasitol 20(7):340-346.<br />
reductase from human erythrocytes. <strong>The</strong> sequence of the<br />
NADPH domain and of the interface domain. Eur J Biochem<br />
Sharma SK and Banyal HS. 2007. Glutathione synthetase in<br />
121,259-267.<br />
Plasmodium berghei. J Parasit Dis 31:33-37.<br />
Krauth-Siegal RL, Muller JG, Lottspeich F and Schirmer RH.<br />
1996. Glutathione reductase and glutamate dehydrogenase<br />
of Plasmodium falciparum, the causative agent of tropical<br />
malaria. Eur J Biochem 235:345-350.<br />
Laemmli UK. 1970. Clevage of structural proteins during the<br />
assembly of the head of bacteriophage T4. Nature 227:680-<br />
685.<br />
Ulusu G, Ciftci M, Erat M, Sakiroglu H and Bakan E. 2005.<br />
Purification and characterization of glutathione reductase<br />
from sheep liver. Turk J Vet Anim Sci 29:1109-1117.<br />
Worthington DJ and Rosemeyer MA. 1974. Human glutathione<br />
reductase: purification of crystalline enzyme from<br />
erythrocytes. Eur J Biol 48:167-177.
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 30-33<br />
Original paper<br />
J P D<br />
Human sparganosis in Sri Lanka: a case report and<br />
review of the cases<br />
R. L. Ihalamulla, H. Gunatilaka and S. D. Fernando<br />
Department of <strong>Parasitology</strong>, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka.<br />
Received 27 February <strong>2008</strong>; revised 22 April <strong>2008</strong>; accepted 15 May <strong>2008</strong><br />
ABSTRACT. <strong>The</strong> occurrence of Spirometra spp. larvae infection is rare in Sri Lanka; only five cases<br />
have been documented so far. Herein, we report a new case of sparganosis in a three year old girl. In all<br />
these cases, there is no history of eating raw flesh of paratenic hosts or application of poultices from<br />
such hosts. Drinking water contaminated with Cyclops, infected with cestode larvae, seems to be the<br />
source of infection in Sri Lanka.<br />
Keywords: sparganosis, Sri Lanka.<br />
INTRODUCTION<br />
Human sparganosis is a zoonotic infection, usually of<br />
subcutaneous tissue, caused by plerocercoids or second<br />
stage larvae (sparganum) of the genus Spirometra<br />
(Pseudophyllidea:Diphyllobothriidae). <strong>The</strong> adults of<br />
these tapeworms are natural intestinal parasites of some<br />
species of domestic and wild canids and felids. <strong>The</strong> life<br />
cycle involves two intermediate hosts. <strong>The</strong> first one is a<br />
freshwater crustacean (copepod) such as Cyclops, and<br />
the second one, which is a paratenic host, belongs to<br />
different kinds of vertebrates (snakes, frogs,<br />
hedgehogs, fowl, pigs and man). Humans are accidental<br />
hosts in whom a subcutaneous nodule, which contains<br />
the larval tapeworm would appear (Chandler, 1955).<br />
<strong>The</strong> surgical removal of worm cures the condition.<br />
However, the infection can cause serious damage when<br />
vital organs such as brain or eye are involved, especially<br />
if larvae have long-lived and are invasive. Human<br />
sparganosis is rare, and about 300 cases have been<br />
reported the world-over (reviewed by Norman et al.,<br />
1980; Kron et al., 1991). <strong>The</strong> majority of the cases have<br />
been reported from the American countries (Norman et<br />
Corresponding author: Dr. S. D. Fernando, Department of<br />
<strong>Parasitology</strong>, Faculty of Medicine, University of Colombo,<br />
Kynsey Road, Colombo 08, Sri Lanka. Email: deepfern@slt.lk<br />
al., 1980; Kron et al., 1991) and the Far East (reviewed<br />
by Ausayakkun et al., 1993; Tsou and Huang, 1993).<br />
Case reports from South Asia are rare (reviewed by<br />
Wijesundera et al., 1997). From Europe, eight cases<br />
have been published (Pampiglione et al., 2003; Rehak<br />
et al., 2006). Herein, we report the sixth case from Sri<br />
Lanka.<br />
Case report: In March 2007, a three year old girl from<br />
the District of Gampaha, in the Western Province of Sri<br />
Lanka, presented at the Base Hospital, Gampaha, with a<br />
one month old lump in the anterior abdominal wall. <strong>The</strong><br />
lump was excised under general anaesthesia. In the<br />
middle of the lump, a worm was found and this was<br />
removed and sent to the Department of <strong>Parasitology</strong>,<br />
Faculty of Medicine, University of Colombo, <strong>for</strong><br />
identification.<br />
Description of the worm: On gross examination the<br />
worm was found to be flat, solid and creamy-white in<br />
colour, and was in three pieces. It showed<br />
pseudosegmentation. <strong>The</strong> anterior or the head-end was<br />
broader (1.2 mm) than the rest of the body (456 µm).<br />
<strong>The</strong>re was a subtle invagination at the anterior margin<br />
of the head. Haematoxylin and eosin (H & E) stained<br />
sections of the body showed a typical cestode cuticle,<br />
subcuticular cells and parenchymatous tissue which
Review of sparganosis in Sri Lanka<br />
31<br />
filled the space with longitudinal muscle bundles surrounded by tracts of suppurative inflammation.<br />
scattered at random (Fig. 1). A few thin walled<br />
excretory ducts were also observed. According to the<br />
<strong>The</strong> second case reported was a 28 year old woman<br />
description given by Chandler, 1955, the worm was<br />
presented with a swelling of the left upper arm of two<br />
identified as a plerocercoid larva (sparganum) of<br />
months duration. <strong>The</strong> lump was non-tender and of 2 cm<br />
Spirometra spp.<br />
diameter. It was excised surgically. H & E stained<br />
sections of the lump showed a cestode larva surrounded<br />
DISCUSSION<br />
by the host-tissue. <strong>The</strong> presence of calcareous<br />
corpuscles, with no concentric layers, suggested that<br />
All the six cases of human sparganosis reported from<br />
this larva was probably a tetrathiridium of a<br />
Sri Lanka involved the subcutaneous tissue, except <strong>for</strong><br />
Mesocestoides worm. Mesocestoides worms have been<br />
a single case of cerebral sparganosis (Alibhoy et al.,<br />
reported from cats and dogs in Sri Lanka (Seneviratna,<br />
2007). <strong>The</strong> first two cases of human sparganosis from<br />
1955; Seneviratna and Mahalingam, 1962).<br />
Sri Lanka caused by plerocercoid larvae were reported<br />
in 1997(Wijesundera et al., 1997). <strong>The</strong> larvae were seen Subsequently, two more cases involving the anterior<br />
in tissue sections of subcutaneous lumps in the left abdominal wall were reported. <strong>The</strong> first of these was<br />
groin and left upper arm, respectively. In the first case, a that of a 40 year old male from the District of Kurunegla<br />
45 year old man from the District of Kegalle was seen at in the north-western province, who presented with a<br />
the General Hospital, Kegalle, with a painless lump in painful tender lump of about five years duration<br />
the groin of 3 months duration. On examination, a non- (Dissanaike et al., 2001). During surgery, a preliminary<br />
tender lump of about 4 cm diameter was seen and an diagnosis of an omental protuberance from<br />
excision biopsy was done. H & E-stained section of the paraumbilical hernia was made by the surgeon. An<br />
lump showed a solid cestode larva that measured 1.1 ovoidal subcutaneous granuloma with a length of 10<br />
mm in width. <strong>The</strong> parasite was in the subcutaneous fat cm, a width of 8.7 cm and a thickness of about 2 cm was<br />
Fig. 1. Section of bodyof larva; bar = 200 µm
32 Ihalamulla et al.<br />
dissected out. From the middle of the granuloma a live<br />
worm crept out. <strong>The</strong>re was no evidence of hernia. <strong>The</strong><br />
worm was a plerocercoid larva (sparganum) of<br />
Spirometra spp, as identified by external morphology<br />
and histological studies.<br />
<strong>The</strong> next case was also reported by Dissanaike et al.,<br />
2001 and involved a young female of 23 years from the<br />
District of Kalutara in the western province. <strong>The</strong> patient<br />
presented with a migratory lump of 4 months duration<br />
with associated pain, in the anterior abdominal wall to<br />
the left of the umbilicus. She also complained of<br />
nausea, fever and loss of appetite <strong>for</strong> the same period.<br />
Surgery of the lump revealed an oval-shaped 3 cm<br />
long and 2 cm wide mass of fibrous and fatty tissue.<br />
When this was opened a whitish, thread like worm<br />
emerged. This worm too was a sparganum of<br />
Spirometra spp., identified in the same way as in the<br />
previous case.<br />
<strong>The</strong> fifth case was that of a 13 year old boy who<br />
presented with three focal motor seizures which<br />
involved the right arm and leg, over a period of three<br />
days (Alibhoy et al., 2007). An enhancing mass lesion<br />
with perilesional oedema in the left frontal lobe was<br />
observed in neuroimaging. <strong>The</strong> patient's blood reports<br />
were normal. Craniotomy and excision of the lesion<br />
was per<strong>for</strong>med. <strong>The</strong> child had remained seizure-free on<br />
carbamazepine therapy since surgery. H & E-stained<br />
sections of the lesion showed a granulomatous reaction<br />
consisting of a necrotic area surrounded by histiocytes,<br />
neutrophils, plasma cells and eosinophils. Sections of a<br />
typical cestode larva showed prominent calcareous<br />
bodies with concentric laminations. This was also<br />
probably a sparganum of Spirometra spp.<br />
<strong>The</strong> adult worms of these tapeworms, which are natural<br />
parasites of cats and dogs, shed eggs which hatch into<br />
coracidia, in water. <strong>The</strong>se eggs, if ingested by<br />
crustaceans such as Cyclops, develop into the<br />
procercoid or the first larval stage. Ingestion of infected<br />
Cyclops by the second intermediate hosts such as frogs,<br />
snakes, fowl and some mammals including humans,<br />
give rise to the second larval stage, the plerocercoid<br />
larva or the sparganum. <strong>The</strong> tapeworm species of<br />
Spirometra (Pseudophyllidae) and Mesocestoides<br />
(Cyclophyllidea) have been reported in dogs and cats in<br />
Sri Lanka (Seneviratna, 1955; Seneviratna and<br />
Mahalingam, 1962). <strong>The</strong> larval stages of these worms,<br />
spargana of Spirometra and tetrathiridia of<br />
Mesocestoides cannot be distinguished, macroscopically.<br />
However, in transverse sections, the headend<br />
of the sparganum shows bothrial slits, whereas that<br />
of the tetrathiridium shows suckers. Further, the<br />
sections of the body show different arrangements in<br />
parenchymal longitudinal musculature. In sparganum,<br />
the bundles of musculature are scattered at random in<br />
the parenchyma, and in tetrathiridium these <strong>for</strong>m an<br />
elliptic band (Andersen, 1983). <strong>The</strong> presence or the<br />
absence of concentric rings in calcareous corpuscles<br />
also helps in differentiating the two larvae; the rings<br />
being absent in the Mesocestoides larvae. In the second<br />
case reported from Sri Lanka (Wijesundera et al.,<br />
1997), the larva had no concentric rings in the<br />
calcareous corpuscles and it was probably a<br />
tetrathiridium. If so, this would be the first report of a<br />
human infection by larva of Mesocestoides spp.<br />
However, it was not possible to confirm the diagnosis as<br />
the scolex of the larvae could not be demonstrated.<br />
Spargana of Spirometra erinacei (=Mansoni) is known<br />
to infect humans in Asia and S. mansonoides in the<br />
Americas. S. erinacei had been reported from dogs in<br />
Sri Lanka (Seneviratna, 1955). <strong>The</strong> human sparganosis<br />
cases reported from Sri Lanka were probably caused by<br />
spargana of S. erinacei.<br />
Humans can rarely be final hosts of Spirometra spp.<br />
<strong>The</strong>y can become infected in three known ways: (a) by<br />
drinking water contaminated with parasitized<br />
copepods, (b) by consuming raw or undercooked meat<br />
of a parasitized second intermediate host, and (c) by<br />
direct entry of plerocercoid larva through the skin or the<br />
mucosa (eye, vagina) or through an open wound, when<br />
poultices made of flesh of intermediate hosts such as<br />
frogs or snakes are applied as local remedies (Miyazaki,<br />
1991). <strong>The</strong> application of poultices is common practice<br />
in Vietnam and Thailand (Ausayakkun et al., 1993;<br />
Miyazaki, 1991). In Sri Lanka flesh of frogs and snakes<br />
is not eaten and any other meat (<strong>for</strong> example pork) is<br />
fully cooked be<strong>for</strong>e eating; poultices made from flesh<br />
are rare. <strong>The</strong>re<strong>for</strong>e, the only possible way humans can<br />
get infected remains by drinking contaminated water.<br />
In the first report (Wijesundera, 1997) the patient had<br />
been drinking water that was not boiled, from an<br />
unprotected well. In the second case report<br />
(Wijesundera, 1997), the patient had a history of<br />
swimming in a pool. It is possible that she accidentally<br />
swallowed some water from the pool. In the present<br />
case, the child visited her aunt's home frequently where<br />
she drank well water that was not boiled.<br />
<strong>The</strong> presence of the natural cestode hosts, namely dogs<br />
and cats as pets is another contributing factor <strong>for</strong> human<br />
infection. When the pets are not regularly dewormed,<br />
they would be harbouring parasites–a ready source of
Review of sparganosis in Sri Lanka<br />
33<br />
infection. <strong>The</strong> man in the first case had two cats and a Andersen KI. 1983. Description of musculature differences in<br />
dog as pets. Unprotected wells are another factor that spargana of Spirometra (Cestoda: Pseudophyllidea) and<br />
tetrathyridia of Mesocestoides (Cestoda; Cyclophyllidea)<br />
should be taken into consideration. Rain water or open<br />
and their value in identification. J Helminth 57:331-334.<br />
drains washing the surrounding surface layers could<br />
bring worm eggs from the soil polluted with host faeces. Ausayakkun S, Siriprasert V et al., 1993. Ocular sparganosis in<br />
This would contaminate the water, which when drunk Thailand. Southeast Asian J Trop Med and Pub Health<br />
24:603-606.<br />
without boiling can bring about the infection.<br />
Chandler C. 1955. <strong>The</strong> Cestoidea or Tapeworms. In: Introduction<br />
<strong>The</strong> early removal of sparganum is important<br />
th<br />
to <strong>Parasitology</strong>. C. Chandler (Ed.) 9 Edition. John Wiley and<br />
considering the facts that some of the larvae could live Sons, Inc., New York. pp 329-374.<br />
<strong>for</strong> over five years, that they have the ability to migrate<br />
Dissanaike AS, Anthonis PR et al., 2001. Two more cases of<br />
inside the body and to becoming invasive, and thus to<br />
sparganosis from Sri Lanka. <strong>The</strong> Ceylon J Med Sci 44:19-22.<br />
cause proliferative sparganosis. Additionally, the larvae<br />
can damage tissues of vital organs leading to serious Holodniy M, Almenoff J et al., 1991. Cerebral sparganosis: case<br />
consequences. Headache and seizures, with or without<br />
report and review. Rev Infect Dis 13:155-159.<br />
focal neurological signs, are common indications of Kron MA, Guderian R et al., 1991. Abdominal Sparganosis in<br />
cerebral sparganosis (Anders et al., 1984). <strong>The</strong> remedy Ecuador: a case report. Am J Trop Med Hyg 44:146-150.<br />
of choice that has been curative <strong>for</strong> both cerebral and Miyazaki I. 1991. Spirometriasis. In: An illustrated book of<br />
subcutaneous sparganosis has been the surgical Helmintic Zoonoses. Miyazaki I. (Ed.) Tokyo: International<br />
excision of the parasite (Holodniy et al., 1991).<br />
Medical Foundation of Japan. pp 207-214.<br />
In Sri Lanka, because raw or undercooked meat is Norman SH and Kreutner A. 1980. Sparganosis: clinical and<br />
pathologic observation in ten cases. Southern Med J 73:297-<br />
seldom if ever consumed and application of poultices<br />
300.<br />
prepared from paratenic hosts is not practiced in<br />
traditional medicine, the simplest way to prevent Pampiglione S. And Fioravanti ML et al., 2003. Human<br />
human sparganosis is to use boiled water <strong>for</strong> drinking sparganosis in Italy. Case report and review of the European<br />
cases. Acta pathologica, microbiologica et immunologica<br />
purposes.<br />
Scandinavica 111:349-354.<br />
ACKNOWLEDGEMENTS Rehak M, Kolarova L et al., 2006. Ocular sparganosis in the<br />
Czech Republic–a case report. Klinick'a mikrobiologie a<br />
We wish to thank Professor A. S. Dissanaike, Emeritus Infekc ni Le'kar stvi 155:161-164.<br />
Professor, Department of <strong>Parasitology</strong>, Faculty of<br />
Medicine, University of Colombo, Sri Lanka, <strong>for</strong> his Seneviratna, P. 1955. A check list of helminths in the Department<br />
of Veterinary Pathology. University of Peradeniya. Ceylon<br />
valuable comments and Ms. R. L. Dewasurendra of the<br />
Vet J 3:32-37.<br />
same Department <strong>for</strong> assistance. We acknowledge the<br />
assistance provided by Mr. R. A. A. N. Ranawaka of the Seneviratna P and Mahalingam S. 1962. Some helminths hitherto<br />
Audio Visual Unit, Faculty of Medicine, University of not reported from Ceylon. Ceylon Vet J 10:88-92.<br />
Colombo and Ms. R. A. D. M. Jayanthi of the Tsou M and Huang TW. 1993. Pathology of Subcutaneous<br />
Postgraduate Institute of Medicine, University of sparganosis: report of two cases. J Formosa Med Assoc 92:<br />
Colombo, <strong>for</strong> microphotography.<br />
649- 653.<br />
REFERENCES<br />
Wijesundera M de S, Ratnatunga N et al., 1997. First reports of<br />
subcutaneous sparganosis in Sri Lanka. Ceylon Med J 42:30-<br />
Alibhoy AT, Fernando R et al., 2007. A case of cerebral 32.<br />
sparganosis. Ceylon Med J 52:93-94.<br />
Anders K, Foley K et al., 1984. Intracranial sparganosis: an<br />
uncommon infection. J Neurosurgery 60:1282-1286.
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 34-41<br />
Original paper<br />
J P D<br />
Immunocytochemical demonstration of 5-hydroxytryptamine<br />
and localization of monoamine oxidase in<br />
Gigantocotyle explanatum and Gastrothylax crumenifer<br />
(Digenea: Paramphistomidae)<br />
1 2 3<br />
S. Ghani , Z. Ghani and S. M. A. Abidi<br />
1<br />
Department of Zoology, Sunbeam College <strong>for</strong> Women, Varanasi.<br />
2<br />
Department of Zoology, A. K. A. P. G. College, Varanasi.<br />
3<br />
Section of <strong>Parasitology</strong>, Department of Zoology, Aligarh Muslim University, Aligarh.<br />
Received 13 March <strong>2008</strong>; revised 27 May <strong>2008</strong>; accepted 30 <strong>June</strong> <strong>2008</strong><br />
ABSTRACT. <strong>The</strong> biogenic amine, 5-hydroxytryptamine (5-HT or serotonin) and its metabolizing<br />
enzyme monoamine oxidase (MAO) were found to be differentially distributed in different regions of<br />
liver and rumen amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer, respectively,<br />
which infect Bubalus bubalis. Strong immunofluorescence, which indicated the presence of 5-HT,<br />
revealed the presence of serotoninergic network in the suckers, pharynx, tegumental and subtegumental<br />
musculature, parenchyma and uterine region of both the amphistomes. Intense<br />
immunoreactivity in the muscular gonopore region was, however, more prominent in G. explanatum.<br />
<strong>The</strong> luminal contents of gut in G. crumenifer also displayed strong reactivity <strong>for</strong> 5-HT, whereas<br />
diffused distribution of this neurotransmitter was observed in the ventral pouch of the rumen<br />
parasite. <strong>The</strong> biogenic amine catabolizing enzyme MAO was also found to be co-distributed with<br />
differential stain intensity in various regions of the two amphistomes. Strong MAO activity was colocalized<br />
in the oral sucker, vitellaria and subtegumental regions of both the parasites, whereas the<br />
enzyme activity was sparsely distributed in gonads. Distinct distribution of MAO was also evident in<br />
the acetabulum region of rumen dwelling worms, whereas in the worms of liver a comparatively weak<br />
enzyme activity was recorded. Such variations in the distribution of 5-HT and MAO could be due to<br />
the differences in the physicochemical nature of the microenvironment as well as feeding preferences<br />
of the host that might exert an influence on the parasites resulting into adaptive variations exhibited<br />
by the amphistome species which infect the same host but occupy different microenvironment.<br />
Keywords: amphistome, histochemical localization, 5-hydroxytryptamine, immune-reactivity,<br />
monoamine oxidase<br />
INTRODUCTION recognized in platyhelminths, among which 5-<br />
hydroxytryptamine (5-HT) appears to be the dominant<br />
A number of neuronal messengers have been well<br />
biogenic amine in all classes (Bennett et al., 1969; Lee<br />
et al., 1978; Fairweather et al., 1987; Maule et al., 1993;<br />
Corresponding author: Dr. S. M. A. Abidi, Section of<br />
<strong>Parasitology</strong>, Department of Zoology,Aligarh Muslim<br />
Halton et al., 1994; Reuter et al., 1995; Mair et al.,<br />
University, Aligarh-202 002, India.<br />
2000; Trim et al., 2001; Stewart et al., 2003; El-Naggar<br />
E-mail: abbasabidi92@hotmail.com
Localization of 5-HT and MAO in amphistomes<br />
35<br />
et al., 2004; 2007). In helminthes, 5-HT has a number of in primary antiserum at a dilution of 1:5 in PBS <strong>for</strong> 18 h<br />
important roles in behavioural and physiological at 4°C. After washing <strong>for</strong> 30 min in PBS, slides were<br />
processes like feeding, locomotion, circadian rhythms, incubated with fluorescine isothyocyante (FITC)-<br />
learning and memory, synaptic growth and plasticity labeled anti-rabbit IgG at a dilution of 1:100 <strong>for</strong> 40 min<br />
(Walker et al., 1996). In addition to its significance in at 4°C. <strong>The</strong> sections were washed again in PBS <strong>for</strong> 90<br />
neurotransmission, 5-HT also exerts an influence on the min, mounted in glycerol and examined under<br />
carbohydrate metabolism, particularly on glycolysis ultraviolet light under an Olympus BH fluorescence<br />
and glycogenolysis in parasites (Rahman and Mettrick, microscope. <strong>The</strong> control slides were processed<br />
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<br />
phosphofructokinase, phosphorylase and stimulates the antiserum.<br />
uptake of glucose (Mansour, 1959a; 1959b; Mansour<br />
and Mansour, 1962; Mansour, 1979; Rahman and<br />
Histoenzymological localization of MAO: For the<br />
Mettrick, 1985). <strong>The</strong> immunoregulatory role of 5-HT<br />
histochemical localization of MAO in G. explanatum<br />
has also been reported in mammalian system, wherein and G. crumenifer, cryocut sections (8 µm thick) were<br />
this amine suppresses the antibody production (Qiu et immediately incubated in the test medium containing<br />
al., 1996). Although the presence of 5-HT has been 4.6 mM tyramine-HCl, 0.1M Tris-HCl, nitro blue<br />
reported in a number of parasitic helminthes; however, tetrazolium (Nitro BT) and distilled water <strong>for</strong> 5 h at<br />
apparently, no report is available on its distribution in 37+1°C (Abidi, 1990). Controls were also run<br />
amphistomes and almost nothing is known about the simultaneously by similarly treating the sections at<br />
rapid inactivation of neurotransmitter like serotonin by 80°C <strong>for</strong> 1 h, or by omitting the substrate from the<br />
catabolizing enzyme, MAO, in amphistomes.<br />
reaction mixture.<br />
<strong>The</strong> monoamine oxidase is known to act as a major RESULTS<br />
scavenger of neuroamines released at the neuronal<br />
Immunoreactivity of 5-HT: <strong>The</strong> results of the IFA test<br />
junctions as well as <strong>for</strong> non–neurotransmitter amines.<br />
revealed differential distribution of 5-HT in G.<br />
<strong>The</strong>re<strong>for</strong>e, in the present study, 5-HT and MAO have<br />
explanatum and G. crumenifer (Table I, Fig. 1–10). <strong>The</strong><br />
been studied immunocytochemically and<br />
tegumental and sub-tegumental regions of both the<br />
histoenzymologically, respectively, in the two<br />
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<br />
amphistomes occupying different micro-environments<br />
immunofluorescence (Fig. 2 and 7), which suggest the<br />
within the same host, Bubalus bubalis.<br />
presence of 5-HT at the host-parasite interface.<br />
MATERIALS AND METHODS<br />
In G. explanatum, moderate immuno-reactivity to 5-HT<br />
Parasite collection: Mature and active flukes of revealed the aminergic network in the oral sucker and<br />
Gigantocotyle explanatum and Gastrothylax muscular pharynx, whereas low level of biogenic amine<br />
crumenifer were collected from the infected liver and appeared to be distributed in the parenchymatous areas,<br />
rumen of eight <strong>Indian</strong> water buffaloes, Bubalus bubalis, thus showing weak immunofluorescence in this region<br />
freshly slaughtered at the local abattoir. <strong>The</strong> worms (Fig. 1–5). <strong>The</strong> distribution of 5-HT appeared to be<br />
were thoroughly washed in glucose-free Hanks' fairly strong around the gonopore, whereas low to<br />
balanced salt saline (HBSS; pH 7.4) at 37+1°C be<strong>for</strong>e moderate distribution was recorded in the uterus and<br />
being processed <strong>for</strong> various studies.<br />
acetabular region.<br />
Immunocytochemical localization of 5-HT: <strong>The</strong> In contrast, the distribution of 5-HT in G. crumenifer<br />
indirect immunofluorescence antibody (IFA) technique was moderate to intense in the intestinal region,<br />
of Coons et al. (1955) and Cable et al. (1996) was whereas the gut contents showed very strong<br />
employed <strong>for</strong> the demonstration of 5-HT. <strong>The</strong> worms immunofluorescence. <strong>The</strong> intestinal muscles and<br />
were embedded in Tissue-Tek II and sectioned at 10 µm<br />
acetabular region exhibited low to moderate activity,<br />
on a Yorco cryotome at -30 °C. <strong>The</strong> sections were<br />
which was also evident in the ventral pouch region.<br />
mounted on glass slides and air-dried <strong>for</strong> 1 h, followed<br />
However, in oral sucker, pharynx, uterus and<br />
by blocking of non-binding sites with blotto (5%<br />
parenchymatous region, weak activity was observed<br />
skimmed milk prepared in phosphate-buffered saline<br />
(Fig. 6–10). Immunofluorescence was completely<br />
(PBS) <strong>for</strong> 4 h. <strong>The</strong>reafter, the sections were washed in<br />
absent from the syncitial layer of the tegument in both<br />
PBS <strong>for</strong> 10 min x 3–4 times at 25°C, and then incubated<br />
the amphistomes under study.
36 Ghaniet al.<br />
Fig. 1–5. Immunocytochemical localization of 5-HT in different regions of liver amphistome, G. explanatum.<br />
Os: oral sucker, Sc: syncitial layer, Stg: sub-tegument, Pa: parenchyma, Go: gonopore, Ph: pharynx, Eg: egg, Ac: acetabulum.<br />
Scale bar = 8 m, magnification x 100<br />
Fig. 6–10. Immunocytochemical localization of 5-HT in different regions of rumen amphistome, G. crumenifer.<br />
Os: oral sucker, Nc: nerve cord, Sc: syncitial layer, Stg: sub-tegument, Pa: parenchyma, Go: gonopore, Ph: pharynx, Eg: egg,<br />
Ic: intestinal caeca Ac: acetabulum, Vp: ventral pouch.<br />
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<br />
37<br />
Fig. 11–18. Histoenzymological localization of monoamine oxidase in different regions of G. explanatum and G. crumenifer.<br />
Os: Oral sucker, Tg: Tegument, Stg: Sub-tegument, Pa: Parenchyma, Vit: Vitellaria, Ic: Intestinal caecae, Im: Intestinal<br />
muscles, Il: Intestinal lumen, Ts: testes, Ov: ovary, Es: egg shell, Vp: ventral pouch, Cm: circular muscles, Ac: acetabulum,<br />
Acm: acetabular muscles. Scale bar = 5 m.(Fig. 11–18)<br />
Magnification = x 100
38 Ghaniet al.<br />
Table I. Immunocytochemical localization of 5-HT in G. explanatum and G. crumenifer<br />
5-HT immunoreactivity in<br />
Worm region G. explanatum G. crumenifer<br />
Syncitium - -<br />
Tegument + + + + + +<br />
Sub- tegument + + / + + + + +<br />
Oral sucker + + +<br />
Pharynx + + +<br />
Intestinal caecae<br />
(i) Intestinal muscles - + / + +<br />
(ii) Intestinal lumen - ++ / + ++<br />
Parenchyma + +<br />
Ventral pouch + +<br />
Gonads<br />
(i) Testis - -<br />
(ii) Ovary - -<br />
Gonopore + ++<br />
Uterus +/ + + +<br />
Acetabulum<br />
(i) Outer lining + / + + + / + +<br />
(ii) Inner lining + / + + + / + +<br />
- absent; + low; + + moderate; + + + intense; + / + + low to moderate; + + / + + + moderate to intense<br />
Table II. Histoenzymological localization of MAO in liver and rumen amphistomes, G. explanatum and G.<br />
crumenifer<br />
Enzyme activity in<br />
Worm region G. explanatum G. crumenifer<br />
Syncitium - -<br />
Sub-tegument + + / + + + + + / + + +<br />
Oral sucker + + + + / + + +<br />
Intestinal caecae<br />
(i) Intestinal muscles + + +<br />
(ii) Intestinal lumen - -<br />
Vitellaria + + + + +<br />
Parenchyma + +<br />
Ventral pouch + + +<br />
Gonads<br />
(i) Testes + + / + + + + + / + + +<br />
(ii) Ovary - / + - / +<br />
Acetabulum<br />
(i) Outer periphery + +<br />
(ii) Middle + + + / + + +<br />
(iii) Inner periphery + +<br />
- absent; + low; + + moderate; + + + intense; - / + absent to low; ++ / + + + moderate to intense<br />
MAO localization: MAO was found to be trematode, whereas intense activity was observed in<br />
differentially distributed, as revealed by the <strong>for</strong>mazan vitellaria. <strong>The</strong> oral sucker and intestinal muscles<br />
deposits, but appeared to be substantially co-localized displayed moderate activity, but weak enzyme activity<br />
in the areas where 5-HT was also found to be distributed was recorded in the parenchymatous and the acetabular<br />
in both the parasites under study (Table II, Fig. 11–18). regions. <strong>The</strong> MAO activity was indistinct in the ovary<br />
Moderate to intense enzyme activity was recorded in and was totally absent from the intestinal lumen (Fig.<br />
the tegument, sub-tegument and testes of the liver 11–14).
Localization of 5-HT and MAO in amphistomes<br />
39<br />
<strong>The</strong> sub-tegumental, oral sucker and testes regions of feeding because in C. elegans the occurrence of 5-HT<br />
G. crumenifer displayed moderate to strong enzyme was found to be necessary <strong>for</strong> stimulating the<br />
activity. <strong>The</strong> localization of MAO in the ventral pouch pharyngeal pumping (Raizen and Avery, 1994; Niacaris<br />
region was also strong. <strong>The</strong> enzyme activity was and Avery, 2003), and the process of ingestion could be<br />
sparsely distributed in the surrounding tissues of the blocked by the serotonin antagonist, gramine (Avery<br />
intestinal caecae. <strong>The</strong> characteristic distribution pattern and Horvitz, 1990). Similarly, the distribution of 5-HT<br />
of MAO could be seen within the acetabular region of has also been demonstrated in the pharyngeal region of<br />
this trematode (Fig. 15–18). other helminthes, G. vitelliloba, F. hepatica and in A.<br />
DISCUSSION<br />
suum (McKay et al., 1991; Brownlee et al., 1994; 1995;<br />
Gustafsson et al., 2001), indicating its role in<br />
<strong>The</strong> present investigation revealed differential neuromuscular coordination.<br />
distribution of serotoninergic network in G.<br />
However, a marked difference in the distribution of 5-<br />
explanatum and G. crumenifer. <strong>The</strong> differential<br />
HT in the intestinal caecae of liver and rumen<br />
immunofluorescence <strong>for</strong> 5-HT was observed in both the<br />
amphistomes was observed. It was totally absent in the<br />
worms, where tegumental and sub-tegumental regions<br />
intestinal caeca of liver amphistome, whereas moderate<br />
displayed moderate to intense immunoreactivity. It is<br />
to intense immunoreactivity was present in the outer<br />
well known that 5-HT plays an important role in<br />
periphery and intestinal lumen of the rumen parasite,<br />
neuromuscular coordination, thus, indicating its<br />
but MAO activity was observed in the sub-mucosal and<br />
potential role in the motility of the two amphistomes as<br />
intestinal muscles of both the parasites. Since about<br />
also reported <strong>for</strong> other digenetic trematodes, S.<br />
90% of the total body content of 5-HT is found within<br />
mansoni, F. hepatica, B. gracilescens and the<br />
the gastrointestinal tract (Erspamer, 1966), there<strong>for</strong>e, it<br />
nematodes, A. tetraptera and A. suum (Anya, 1973;<br />
is possible that 5-HT of host origin may also be<br />
Fairweather et al., 1984; 1987; Mair et al., 2000; Trim<br />
absorbed via the alimentary route in G. crumenifer, as<br />
et al., 2001; Stewart et al., 2003). <strong>The</strong>se results were<br />
suggested by Martin and Donahue (1989) <strong>for</strong> A. suum<br />
further augmented by the co-distribution of strong<br />
that 5-HT can be absorbed by the gastrointestinal<br />
MAO activity in sub-tegumental region. Adults and<br />
epithelial cells, there<strong>for</strong>e, the presence of strong<br />
developmental stages of parasitic flatworms rely on<br />
immunofluorescence <strong>for</strong> 5-HT in the lumen of rumen<br />
well-developed neuromuscular system <strong>for</strong> host<br />
amphistome is not surprising. However, the distribution<br />
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<br />
of MAO in close association with gastrointestinal tract<br />
immunoreactivity <strong>for</strong> 5-HT in the selective regions of<br />
could provide a detoxification mechanism to protect the<br />
oral sucker revealed a rich supply of serotoninergic<br />
parasite from any deleterious effects of excess amount<br />
fibres in the well developed muscular organ of the<br />
of biogenic amines of host origin.<br />
amphistomes. However, 5-HT was sparsely distributed<br />
in the acetabular region, whereas strong MAO activity <strong>The</strong> presence of 5-HT and MAO in the parenchymal<br />
could be localized in the peripheral region, close to the region of both the amphistomes and particularly in the<br />
attachment surface area. It has been suggested that muscular lining of ventral pouch of G. crumenifer could<br />
MAO could also participate at the host-parasite be due to the presence of mitochondria, which are<br />
interface in the inactivation of exogenous amines which known to occur along the lining of ventral pouch and in<br />
come from dietary intake by the host (Barrett, 1981). the parenchymal tissues.<br />
Moreno and Barrett (1979) suggested that in H.<br />
diminuta the main function of monoamine oxidase is to<br />
<strong>The</strong> serotoninergic component of neuromuscular<br />
protect the parasite against exogenous amines present<br />
coordination has been reported in the reproductive<br />
in the host alimentary tract, where bacteria also<br />
system of F. hepatica, Moniezia expansa, D. sagittata,<br />
synthesize biogenic amines (Nag et al., 1978).<br />
and H. medioplexus by Fairweather et al., 1987; Maule<br />
<strong>The</strong>re<strong>for</strong>e, MAO can be considered as an important<br />
et al., 1993; Cable et al., 1996; Humphries et al., 1997,<br />
component at the interface detoxifying the excess<br />
respectively. Similarly, the strong immunoreactivity <strong>for</strong><br />
amines in order to maintain a successful host-parasite<br />
5-HT in the gonopore region of G. explanatum and in<br />
relationship.<br />
the uterine musculature of both the flukes suggests the<br />
role of 5-HT in reproductive physiology of the<br />
<strong>The</strong> 5-HT immunoreactivity in the pharynx of both the amphistomes, where 5-HT, beside other putative neuroamphistomes<br />
indicate serotoninergic regulation of transmitters, might be partially involved in vigorous<br />
pharyngeal musculature, thus suggesting its role in contractions of uterine wall during the shaping and
40 Ghaniet al.<br />
passage of eggs, but the higher activity in G. Anya AO. 1973. <strong>The</strong> distribution and possible<br />
explanatum could possibly be due to the fact that neurophamacological significance of serotonin (5-<br />
hydroxytryptamine) in Aspicularis tetraptera (Nematoda).<br />
enormous egg production continues through out the<br />
Comp Gen Pharmacol 4:149-156.<br />
year in this parasite, whereas strict seasonal<br />
reproductive cycle is operative in rumen amphistome, Avery L and Horvitz HR. 1990. Effects of starvation and<br />
G. crumenifer (Hanna et al.,1988). <strong>The</strong>re<strong>for</strong>e, it could neuroactive drugs on feeding in Caenorhabditis elegans. J<br />
Exp Zool 253:263-270.<br />
be possible that any chemotherapeutic intervention in<br />
the metabolism of 5-HT could influence the egg Barrett J. 1981. Biochemistry of Parasitic Helminthes. London<br />
producing activity of these parasites. <strong>The</strong> presence of and Basingstoke, Macmillan publishers Ltd.<br />
monoamine oxidase in gonads is consistent with the Bennett JL, Bueding E, Timms AR and Engstrom RG. 1969.<br />
known transmitter and hormonal role of 5-HT in Occurrence and levels of 5-hydroxytryptamine in<br />
reproductive activity of trematodes. <strong>The</strong> MAO activity Schistosoma mansoni. Mole Pharmacol 5:542-545.<br />
has also been reported in the gonads of female A. galli<br />
Brownlee DJA, Fairweather I, Johnston CF and Shaw C. 1994.<br />
and S. cervi (Mishra et al., 1983; Agarwal et al., 1990). Immunocytochemical demonstration of peptidergic and<br />
In the present study the absence of fluorescence <strong>for</strong> 5- serotoninergic components in the enteric nervous system of<br />
HT in the ovary and testes but the presence of MAO in the round worm Ascaris suum (Nematoda; Ascaroidea).<br />
these regions is of some curiosity. It is possible that this Parasitol 108:89-103.<br />
biogenic amine could be present in the gonads but the Brownlee DJA, Holden-Dye L, Fairweather I and Walker RJ.<br />
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methods used in the present study. <strong>The</strong> KSAYMRF amide on the pharyngeal muscle of the parasitic<br />
immunoreactivity in the eggshells of only G. nematode, Ascaris suum. Parasitol 3:379-384.<br />
explanatum could possibly be due to auto-fluorescence, Cable J, Marks NJ, Halton DW, Shaw C, Johnston CF, Tinsley<br />
which was totally absent in case of G. crumenifer.<br />
RC and Gannicott AM. 1996. Cholinergic, serotoninergic and<br />
peptidergic components of the nervous system of Discocotyle<br />
It is concluded that 5-HT, in concurrence with MAO, is sagittata (Monogenea: polyopisthocotylea). International<br />
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varies in different regions with some specific<br />
Coons AH, Leduc EH and Connolly JM. 1955. Studies on<br />
differences in parasites occupying different antibody production: I. A method <strong>for</strong> the histochemical<br />
microenvironments. <strong>The</strong> pronounced localization of demonstration of specific antibody and its application to a<br />
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acetabular region of the rumen amphistome suggests<br />
El-Naggar MM, Arafa SZ, El-Abbassy SA, Stewart MT and<br />
the involvement of this enzyme at the interface <strong>for</strong> the Halton DW. 2004. Neuromusculature of Macrogyrodactylus<br />
establishment of host-parasite relationship.<br />
clarii, a monogenean gill parasite of the Nile cat fish Clarias<br />
ACKNOWLEDGEMENTS<br />
gariepinus in Egypt. Parasitol Res 94:163-175.<br />
El-Naggar MM, Arafa SZ, El-Abbassy SA, Stewart MT and<br />
<strong>The</strong> authors are thankful to the Chairman, Department Halton DW. 2007. Neuromusculature of Macrogyrodactylus<br />
of Zoology, Aligarh Muslim University, Aligarh, and congolensis, a monogenean skin parasite of the Nile cat fish<br />
Clarias gariepinus. Parasitol Res100:265-279.<br />
Dr. Haris M. Khan of J. N. Medical College, Aligarh,<br />
<strong>for</strong> providing the necessary laboratory facilities, and to Erspamer V. 1966. Occurrence of indole alkylamines in nature.<br />
Dr. M. Khalid Saifullah <strong>for</strong> his comments and Mr. In: Handbook of Experimental Pharmacology. Erspamer V.<br />
Ramesh Chandra <strong>for</strong> parasite collection. Financial (Ed.), Springer, Berlin, Heidelberg, New York. pp 132-181.<br />
Assistance from University Grants Commission, New Fairweather I, Holmes SD and Threadgold LT. 1984. Fasciola<br />
Delhi, and Council of Science and Technology, U. P, hepatica motility response to fasciolicides in vitro. Exp<br />
Lucknow, is gratefully acknowledged.<br />
Parasitol 57:209-224.<br />
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dinucleotide phosphate-diaphorase activity, serotonin Maule AG, Halton DW, Shaw C and Johnston CF. 1993. <strong>The</strong><br />
immunoreactivity and GYIRF amide immunoreactivity and cholinergic, serotoninergic and peptidergic components of<br />
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Halton DW, Maule AG, Brennan GP, Shaw C, Stoitsova SR and McKay DM, Halton DW, Johnston CF, Fairweather I and Shaw<br />
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Hanna REB, Williamson DS, Mattison RG and Nizami WA. Mishra SK, Sen R and Ghatak S. 1983. Monoamine oxidase in<br />
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Moreno MS and Barrett J. 1979. Monoamine oxidase in adult<br />
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Humphries JE, Halton DW, Johnston RN, Maule AG, Johnston<br />
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CF and Shaw C. 1997. Cholinergic, serotoninergic and<br />
Ind J Exp Biol 16:174-176.<br />
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Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 42-46<br />
Original paper<br />
Seasonal dynamics in lungworm infections in sheep<br />
J P D<br />
N. Moghaddar, S. S. Shekar<strong>for</strong>oush and A. Afrahi<br />
School of Veterinary Medicine, Shiraz University, Shiraz, Islamic Republic of Iran<br />
Received 29 January <strong>2008</strong>; revised 31 March <strong>2008</strong>; re-revised 26 May <strong>2008</strong>; accepted 2 August <strong>2008</strong><br />
ABSTRACT. Out of 1804 lungs of sheep examined <strong>for</strong> lungworms during 2001-2005 in abattoirs of<br />
Shiraz, Iran, 529 (29.3 %) of them were found positive. Majority (67.0%) of the lungs had mixed<br />
infection with more than one lungworm species. Seasonal analysis of the occurrence of infection<br />
revealed a significantly high (p > 0.1) incidence in spring (38.5 %) and summer (34.5 %), followed<br />
by autumn (16.7%) and winter (8 %). Muellerius capillaries (51.46 %) and Dictyocaulus filaria<br />
(31.58%) were the predominant worm species, whereas Protostrongylus rufescens (12.54%) and<br />
Cystocaulus ocreatus (4.42%) were relatively less common. Coprological examination<br />
demonstrated a significantly high (p > 0.1) incidence of infection in sheep kept by tribal people<br />
(nomadic people) than those by local ones, as shown by higher intensity of infection and clinical<br />
signs in the tribal animals. Bronchitis, pneumonia with intense damage of parenchyma, and<br />
presence of a large number of nodules of variable size resulted in the rejection of infected lungs.<br />
Effective control measures are suggested.<br />
Keywords: lungworms, Iran, seasonal dynamics, sheep, tribal people<br />
INTRODUCTION<br />
MATERIALS AND METHODS<br />
All over the world, lungworm infections in sheep have Out of a total 2224 sheep included in this study, 978<br />
been well recognized (Rose, 1965; Sammarrae and belonged to the local farmers around Shiraz, the<br />
Sewell, 1977; Dhar, 1985), and have also been capital of Fars Province of Iran, and 1246 to the tribal<br />
reported from Iran (Eslami et al., 1980; Moghaddar et people. <strong>The</strong> observations were recorded in all seasons<br />
al., 1991). However, apparently, no systematic studies throughout the year, depending upon the availability<br />
on seasonal incidence of lungworm infections in of animals. <strong>The</strong> tribal people raised and purchased the<br />
Iranian sheep have so far been made. <strong>The</strong> present animals from different agro-climatic zones, and<br />
investigations were, there<strong>for</strong>e, aimed to study the moved with them in spring and summer in search of<br />
seasonal dynamics of the infections caused by grazing places and suitable markets <strong>for</strong> sale. <strong>The</strong><br />
different lungworms species and their clinico animals were assigned to two groups viz. Group-I:<br />
pathological implications in sheep of local farmers twenty sheep belonging to tribal people in each<br />
and tribal people (nomadic people) in Fars Province of season except in winter (total 180) and twenty from<br />
Iran.<br />
local farmers in each season (total 240), which<br />
showed respiratory distress were selected during<br />
2003-2005 <strong>for</strong> clinical and coprological examinations<br />
and the detailed clinical examination of the selected<br />
animals was done afterwards, Group-II: local<br />
Corresponding author: Dr. N. Moghaddar, School of Veterinary animals and those belonging to tribal people (total<br />
Medicine, Shiraz University, Shiraz, Islamic Republic of Iran.<br />
1804) were also examined in abattoirs of Shiraz during<br />
E-mail: nasrinmoghaddar@yahoo.com<br />
2001-2005.
Lungworm infections in sheep<br />
43<br />
For coprological examination, the faecal samples significantly (p > 0.1) high incidence was observed in<br />
from individual animal were collected per rectum in spring (38.5 %) and summer (34.5 %), as compared to<br />
suitable containers, and examined afresh as far as autumn (16.7 %) and winter (8.0%). Majority (57.6 %)<br />
possible. Of these, a few positive samples were used of the specimens showed low grade infection, whereas<br />
<strong>for</strong> the culture of larvae. <strong>The</strong> samples collected from 67.0 % had mixed infection with more than one<br />
remote areas were preserved in 10% buffered species. <strong>The</strong> species recovered were Muellerius<br />
<strong>for</strong>malin, and then taken to the laboratory. Faecal capillaries (51.46%), Dictyocaulus filaria (31.58%),<br />
examination was per<strong>for</strong>med by direct smear method, Protostrongylus rufescens (12.54%) and Cystocaulus<br />
and the faecal cultures were raised from fresh, positive ocreatus (4.42%), but M. capillaris and D. filaria<br />
faecal samples in petri plates/glass jars depending predominated in both local animals and animals of<br />
upon the faecal quantity (Soulsby, 1982). <strong>The</strong> tribal people. <strong>The</strong> faecal examination revealed (Table<br />
lungworms' larvae from faecal cultures were collected II; Fig. 2) 31.7% and 30.0 % infection during spring<br />
by Baermann technique (Soulsby, loc.cit). Necropsy and summer seasons, respectively, followed by<br />
examination in abattoirs was carried out by examining 12.5% and 11.6 % infection in autumn and winter,<br />
the intact lungs <strong>for</strong> gross lesions and after incision <strong>for</strong> respectively. Animals, which belonged to tribal people<br />
lesions in parenchyma and bronchi. <strong>The</strong> worms were showed significantly (p > 0.1) high infection as<br />
collected from different locations of lungs, washed in compared to the local ones; however, the <strong>for</strong>mer could<br />
physiological saline and collected in tubes. Pieces of not be examined during winter due to their nonlungs<br />
were also used in Baermann apparatus to isolate availability.<br />
the lungworms. Alternatively, the lung pieces were<br />
Gross examination of the lungs showed variable<br />
also placed in warm saline and isolated by<br />
lesions depending upon the worms' species involved,<br />
sedimentation and slow centrifugation. <strong>The</strong><br />
their number and the duration of infection. Severely<br />
worms/larval stages thus recovered were counted by<br />
affected lungs were highly congested and enlarged. At<br />
dilution technique. <strong>The</strong> worms and the juveniles were<br />
places, the organs were hard to touch, while at other<br />
fixed and preserved in 5 or 10 % <strong>for</strong>mol saline <strong>for</strong><br />
places showed firm, raised dark grayish or palefurther<br />
processing and identification. <strong>The</strong> data were<br />
yellow nodules of different size. On incision, the<br />
analysed statistically by Chi-square test of<br />
bronchi contained worms and large amount of mucus<br />
significance.<br />
and exudate mixed with blood. Adult D. filaria and P.<br />
RESULTS rufescens worms were observed to block air<br />
passages. <strong>The</strong> nodules when present, were<br />
Out of 1804 sheep lung specimens examined, 529<br />
circumscribed or enlarged, embedded in the lung<br />
(29.3%) were positive with incidence ranging from<br />
parenchyma and contained spirally coiled adult M.<br />
5.7% to 48.0 % in different seasons (Table I; Fig. 1). A<br />
Table I. Seasonal incidence of lungworms of sheep recorded in abattoirs of Shiraz, Iran<br />
Year of<br />
Seasons of the year<br />
examination Spring Summer Autmn Winter<br />
T +ve %ve T +ve %ve T +ve %ve T +ve %ve<br />
2001 75 36 48.0 222 84 37.8 60 19 31.6 45 3 6.6<br />
2002 95 31 32.6 146 44 30.1 66 8 12.1 25 2 8.0<br />
2003 101 42 41.5 155 49 31.6 75 13 17.3 45 4 8.9<br />
2004 93 25 26.8 120 53 44.1 73 9 12.3 35 2 5.7<br />
2005 85 39 45.8 135 39 28.9 79 20 25.3 74 7 9.4<br />
Total 449 173 38.5* 778 269 34.5* 353 69 16.7 224 18 8.0<br />
Grand total 1804 529 29.3 %<br />
*Significant difference: (p > 0.1) ; T = number of animals examined ; +ve = positive ;<br />
% ve = percent positive.
44 Moghaddar et al.<br />
Table II. Seasonal incidence of lungworms in sheep by faecal examination in Shiraz, Iran<br />
Animals /<br />
Year of<br />
Seasons of the year<br />
Examination Spring Summer Autumn Winter<br />
+ve % +ve % +ve % +ve %<br />
Tribal animals<br />
2003 8 - 6 - 4 - N -<br />
2004 10 - 11 - 3 - N -<br />
2005 9 - 12 - 4 - N -<br />
Total 27 45.0* 29 48.3* 11 18.3 0.0 0.0<br />
_________ ______________ ___________ _____________<br />
Local animals<br />
2003 2 - 3 - 2 - 1 -<br />
2004 5 - 2 - 0 - 3 -<br />
2005 4 - 2 - 2 - 3 -<br />
Total 11 18.3 7 11.6 4 6.6 7 11.6<br />
___________ ______________ ______________ _____________<br />
Grand total (120) 38 31.7* (120) 36 30.0* (120)15 12.5 (60)7 11.6<br />
No. of sheep examined in each season = 20<br />
*Significant difference (p > 0.1) ; + ve = animals positive ; % = percent ; N = nil (not examined);<br />
Figures in parenthesis indicate total no. of animals examined.<br />
capillaries. On few occasions, C. ocreatus (4.42%)<br />
were also recovered. In moderate infections (50-100<br />
worms), the organs were less affected, whereas the<br />
60<br />
50<br />
low-grade infection (less than 50 worms) showed no<br />
perceptible characteristic lesions in the lungs.<br />
Periodic clinical examination of local and tribal's<br />
2001<br />
2002<br />
2003<br />
Percent positive<br />
40<br />
30<br />
20<br />
38.5<br />
34.6<br />
19.5<br />
2004<br />
2005<br />
Total<br />
10<br />
8<br />
0<br />
Spring Summer Autumn Winter<br />
Seasons of the year<br />
Fig. 1. Seasonal incidence of lungworms of sheep recorded in abattoirs of Shiraz, Iran.
Lungworm infections in sheep<br />
45<br />
Percent positive<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Spring Summer<br />
2003<br />
2004<br />
Seasons of year<br />
Tribal's sheep<br />
2005<br />
Local sheep<br />
Autumn Winter Spring Summer AutumnWinter Spring Summer Autumn Winter<br />
Fig. 2. Seasonal incidence of lungworms in tribal's and local sheep by faecal examination in Shiraz, Iran.<br />
sheep infected with lungworms, showed variable and goats in Himalayan region of India. Except <strong>for</strong><br />
symptoms depending upon the intensity of infection. few sporadic reports on the occurrence of lungworms<br />
<strong>The</strong> animals in carrier stage exhibited no marked in sheep in certain parts of Iran, no ef<strong>for</strong>ts were yet<br />
symptoms except <strong>for</strong> slight nasal discharge. In made to investigate the incidence and other aspects of<br />
moderate infections, the symptoms were nasal these important parasites particularly in the animals<br />
discharges, coughing, dyspnea and bronchitis. of tribal people though these animals are a major<br />
However, in heavy infections, the animals showed source of mutton in this region. <strong>The</strong> present<br />
profuse nasal discharges mixed with blood and mucus, investigations, carried out <strong>for</strong> five years <strong>for</strong> the first<br />
uncontrolled coughing, dyspnea, emaciation, time in this country, envisaged the need to evolve and<br />
bronchitis and pneumonia. Few animals showed implement suitable control measures against these<br />
raised body temperature.<br />
parasites, and to study the epidemiology of this<br />
DISCUSSION<br />
infection in animals belonging to tribal people.<br />
<strong>The</strong> observations on the gross lesions caused by<br />
<strong>The</strong> present study established the endemicity of Fars<br />
lungworm species in sheep, in the course of present<br />
Province of Iran <strong>for</strong> lungworm infections. <strong>The</strong><br />
study, corroborated with the earlier reports of<br />
incidence was significantly (p > 0.1) high during<br />
Moghaddar et al. (1991 ) and those of Nimmo (1979),<br />
spring and summer seasons in all categories of the<br />
Pybus and Shave (1984), and Oryan and Moghaddar<br />
animals but the animals of tribal people showed<br />
(1991). However, the association of P. rufescens and<br />
comparatively higher rate of infection. <strong>The</strong>se results<br />
C. ocreatus with the observed extra-nodular lung<br />
were more or less in agreement with the solitary report<br />
lesions was interesting.<br />
of Moghaddar et al. (1991) who observed a quite high<br />
(52.46%) incidence of this infection in sheep during ACKNOWLEDGEMENT<br />
six months of their study between April and September<br />
<strong>The</strong> authors thank the Vice-chancellor, Research <strong>for</strong><br />
in Shiraz area. Earlier reports of Eslami and<br />
financial support.<br />
Monasheri (1978) and Eslami et al. (1980) on the<br />
prevalence of lungworms in sheep from Tehran and REFERENCES<br />
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Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 47-55<br />
Original paper<br />
J P D<br />
Focus on pathogenic trematode cercariae infecting fresh<br />
water snails (Mollusca: Gastrapoda ) of tribal region of<br />
southern Rajasthan, India<br />
S. L. Choubisa<br />
Department of Zoology, Government Post Graduation College, Sirohi.<br />
Received 12 November 2007; accepted 5 September <strong>2008</strong><br />
ABSTRACT. More than 15,000 freshwater snail species, which belonged to Lymnaeidae,<br />
Planorbidae, Viviparidae and Thiaridae families of the order Mollusca were examined <strong>for</strong> the<br />
presence of pathogenic trematode cercariae infection. Lentic (stagnate waters) and lotic (running<br />
waters) habitats and dwelling behaviours of these snails determined their propensity <strong>for</strong> cercariae<br />
infection. In the lentic water, the infection was maximum in the surface dwellers and minimum in the<br />
bottom dwellers. In the lotic habitats, in bottom dwelling snails the cercarial infection was nominal.<br />
From these snail species, a rich diversity of cercariae was observed and six different types of cercariae<br />
viz. amphistome, echinostome, furcocercous, gymnocephalous, monostome and xiphidiocercariae<br />
were found. Few snail species also revealed infection with metacercariae of Strigied, Ophisthorchiide<br />
and Aspidogaster species. <strong>The</strong> most favorable season <strong>for</strong> cercarial infection was found in the late rainy<br />
or pre-winter season, where > 95% mature snails released different kinds of cercariae . In the morning<br />
times, maximum number of cercariae emerged out from their snail hosts. <strong>The</strong>se cercariae larvae,<br />
having pigmented eyes on their body, are very much sensitive to light and are active swimmers. A<br />
strong host-specificity was observed in echinostome, gymnocephalous, furcocercous and monostome<br />
cercariae. In the present communication, the diversity of snails of lentic and lotic habitats, and their<br />
pathogenic trematode larvae (cercariae), seasonal occurrence, behaviours and host-specificity of<br />
various types of cercariae are discussed.<br />
Keywords: cercaria, lentic and lotic habitats, pathogen, Rajasthan, snail hosts, trematodiasis<br />
INTRODUCTION<br />
<strong>The</strong> digentic trematode cercaria larva is an infective and<br />
active stage of the development process of trematode<br />
parasites of man and animals (vertebrates), and these<br />
larvae are also very much pathogenic to their snail hosts<br />
(Erasmus, 1972; Cheng, 1973; Choubisa, 1986a).<br />
Additionally, snails are ideal bio-indicators not only <strong>for</strong><br />
paleo-environments and water quality (Harman, 1974;<br />
Corresponding author: Dr. S. L. Choubisa, 205, Chanakyapuri,<br />
Sector-4, Hiran Magri, Udaipur -313002.<br />
E-mail: choubisasl@yahoo.com<br />
Clarke, 1979), but <strong>for</strong> lotic and lentic aquatic<br />
ecosystems as well (Choubisa, 1992). Hence, many<br />
workers have reported the diversity of the snails of<br />
lentic habitats and their pathogenic cercarial fauna from<br />
different geographical areas (Pandey and Agrawal,<br />
1978; Choubisa and Sharma, 1983). From Rajasthan,<br />
only a few studies have been conducted (Choubisa,<br />
1986b, 1991), although southern Rajasthan has a<br />
number of perennial lentic and lotic habitats. <strong>The</strong>re<strong>for</strong>e,<br />
the present study was undertaken to find out the<br />
diversity of snail hosts inhabiting lentic and lotic<br />
habitats of southern Rajasthan as well as their
48<br />
Choubisa<br />
pathogenic cercariae, besides the study of cercarial<br />
behaviours, host-specificity and their seasonality.<br />
MATERIAL AND METHODS<br />
More than 15,000 live adult snails (gastropods), which<br />
inhabited perennial lentic and lotic habitats of tribal<br />
region (Banswara, Dungarpur, Chittorgarh, Rajsamand<br />
and Udaipur districts) of southern Rajasthan, were<br />
collected and were maintained separately in laboratory<br />
aquaria according to their species and habitats. <strong>The</strong>se<br />
snails were identified according to Ray and Mukherjee<br />
(1963) and Tonapi (1980). <strong>The</strong> snails were exposed to<br />
light <strong>for</strong> the determination of cercariae infection, and<br />
were dissected out to establish the presence of<br />
trematode larvae. <strong>The</strong> larval trematodes were collected<br />
and examined according to the methods reported by<br />
Mukharjee (1980). Thus, the larvae were studied under<br />
light cover-slip pressure, in both alive and stained<br />
mounts. Neutral red and methylene blue stains were<br />
used <strong>for</strong> the detailed study of penetration glands, gut<br />
and genital rudiments. Unstained live cercariae were<br />
studied specially <strong>for</strong> their flame cell <strong>for</strong>mulae. <strong>The</strong>y<br />
were then identified following the key as given by<br />
Erasmus (1972).<br />
For the oberservation of behaviours and swimming<br />
activity of naturally emerging cercariae, infected snails<br />
were kept in separate beaker (500 ml) containing tap<br />
water. For phototactic study, beaker containing infected<br />
snails was covered with a black paper having few small<br />
apertures at mid level of beaker, and then electric bulb<br />
light was passed through these apertures. This<br />
experiment was done in morning in a dark-room.<br />
Simultaneously, encytorment, geotaxism and<br />
swimming activity of different cercariae were also<br />
observed. <strong>The</strong> thigmotaxism behaviour was observed<br />
by contact using a needle.<br />
RESULTS<br />
Diversity of snails and their trematode larvae: From<br />
the perennial lentic and lotic habitats of tribal region of<br />
southern Rajasthan, 15 species of snails (Palte 1,<br />
Fig.1–14) were recovered. Of these, seven species<br />
belonged to Lymnaeidae, two to Planorbidae, two to<br />
viviparidae, and four to Thiaridae (Mellaniidae) family<br />
(Table I). Except the snails of Lymnaeidae family,<br />
others were found to be bottom dwellers. From these<br />
snail species, only one species, Malania (Plotia) scabra<br />
was found to be stenotopic and restricted to the running<br />
waters only.<br />
From these snail species, basically, six types of<br />
Plate 1, Fig. 1–14.<br />
1. Lymnaea acuminata f. patuta<br />
2. L. acuminata f. chlamys<br />
3. L. acuminata f. typical<br />
4. L. luteola f. asutralis<br />
5. L. luteola f. typical<br />
6. L. luteola f. impure<br />
7. L. luteola f. rufescens<br />
8. Indoplanorbis exustus<br />
9. Faunus ater<br />
10. Melania (Plotia) scabra<br />
11. Thiara (Tarebia) lineata<br />
12. M. striatella tuberculata<br />
13. Vivipara bengalensis race gigantea and<br />
14. V. bengalensis race mandiensis<br />
cercarial larvae viz. amphistome, echinostome,<br />
furcoercous, gymnocephalous, monostome and<br />
xiphidocercariae were recovered. Those snail species<br />
having surface dwelling habit (genera of Lymnaeidae<br />
family), showed maximum diversity of trematode<br />
cercarial larvae.
Pathogenic trematode cercariae infecting freshwater snails in Rajasthan<br />
49<br />
Vivipara bengalensis<br />
Cercaria beaveri<br />
C. komiyai<br />
C. thapari<br />
C. pyri<strong>for</strong>mis<br />
C. milleri<br />
C. sewelli C. udaipuriensis<br />
Fig. 15. Different cercarial species which emerged from Vivipara snail species.<br />
However, metacercarial larvae such as plagiorchid and<br />
echinostome from Vivipara bengalensis, strigeid<br />
(Tetracotyle lymnaei) from Lymnaea acuminata<br />
species, aspidogaster from Melania striatella<br />
tuberculata, and opsthorchiid from Indoplanorbis<br />
exustus snail hosts were also traced out. Different types<br />
and species of cercariae and metacercariae from a<br />
common snail species have been shown in Fig. 15–19.<br />
At the same time, two different types of cercariae<br />
(double infection) in a single snail specimen have also<br />
been observed. In general, xiphidio cercariae were<br />
associated with other types of cercariae (furcocercous,<br />
gymnocephalous, monotome and echinostome).<br />
However, such types of double infections were rarely<br />
observed.<br />
Seasonality and periodicity: In general, amphistome,<br />
echinostome, furcocercous, gymnocephalous,<br />
monostome and xiphidio cercariae were found in all<br />
seasons, but their infection % were found to vary<br />
greatly. However, the maximum infection of cercariae<br />
in their respective snail host was found only in the late<br />
rainy or pre-winter season (Table II). But monostome
50<br />
Choubisa<br />
Melanoides tuberculatus<br />
Cercaria tali<br />
C. buckleyi<br />
C. chauhani<br />
C. srivastavi<br />
C. trioculata<br />
C. martini<br />
C. diglandulata<br />
C. chengi<br />
A spidogaster sp.<br />
(metacecaria)<br />
Fig. 16. Different cercarial and matacercarial species which emerged from Melania snail species.<br />
and xiphidio cercariae showed two peak periods of<br />
infection viz., pre-winter and summer seasons. <strong>The</strong><br />
emergence of these trematode larvae from their snail<br />
hosts was observed to be at peak in mornings, nominal<br />
during noon and lowest in evening hours. However,<br />
during night, furco and monostome cercariae also<br />
emerged out but their numbers were relatively very low.<br />
Host-specificity and behaviours: In the present study,<br />
most of the cercariae showed stern host–specificity:<br />
amphistome cercariae were released from genera of<br />
Planorbidae family, echinostome cercariae from genera<br />
of Lymnaedae family and monostome cercariae from<br />
genera of Thiaridae family. But furcocercous and<br />
xiphidio cercariae showed less host-specificity, and<br />
were found with various incidences in almost all the<br />
genera of each family (Table I).<br />
Except furcocercous and monostome cercariae, other<br />
cercariae were generally encysted after 10-30 min, in a<br />
beaker containing tap water. At the resting stage, all<br />
types of cercariae moved-like a caterpillar on the<br />
bottom of beaker but in the active (swimming) state,<br />
they move like a spermatozoa and their heads remained<br />
towards up side. Only furcocercous cercariae showed<br />
jerking movement and these are relatively very active<br />
swimmers having strong phototropic, negative<br />
geotaxic and thigmotaxic behaviours; monostome<br />
cercariae also showed such behaviours (Table III). <strong>The</strong><br />
strongest phototrophic behaviour (maximum
Pathogenic trematode cercariae infecting freshwater snails in Rajasthan<br />
51<br />
Indoplanorbis excustus<br />
Cercaria manteri C. pandei C. dalibaghensis<br />
C. chauhani<br />
Gyraulus convexiusculus<br />
C. chungathi<br />
Fig. 17. Different cercarial species which emerged from genera of Planorbidae snail family.<br />
their habitats were healthy and non-polluted, and had<br />
abundant food. In the southern Rajasthan, most of the<br />
lentic and lotic aquatic habitats are perennial and non-<br />
polluted, containing aquatic vegetation which provided<br />
a healthy environment or ecosystem <strong>for</strong> the breeding<br />
and increase in the population of different snail species.<br />
On the basis of present survey, it was found that the<br />
larval digenean (cercariae) infection in surface<br />
dwelling snails in the lentic habitats, especially in the<br />
perennial ponds or reservoirs, was higher than those of<br />
bottom dwellers in the same habitat. <strong>The</strong> bottom t al.,<br />
emergence and aggregation of cercariae towards light<br />
source) was found in those cercariae, who had eye<br />
pigments on their body such as in the amphistome,<br />
furcocercous and monostome cercariae. In general,<br />
cercariae having large-sized body (except<br />
furcocercous) were passive swimmers. Behaviours of<br />
different cercariae have been hown in Table III.<br />
DISCUSSION<br />
In the present study, a rich diversity of snail species<br />
(intermediate hosts) was found which indicated that
52<br />
Choubisa<br />
Lymnoea luteola<br />
Lymnaea acuminata<br />
Tetracotyle lymnaei<br />
Cercaria indicae LXXIV Echinostome sp. Cercaria VII kerala<br />
C. itoi<br />
Cercaria talensis<br />
Fig. 18. Different cercarial species which emerged<br />
from L. luteola snail species.<br />
Fig. 19. Different cercarial and matacercarial species<br />
which emerged from L. acuminita snail species.<br />
dwellers of the lentic habitats showed greater infection,<br />
as compared to their counter-parts in the lotic habitats.<br />
This has occurred probably because the chances of<br />
contact between free-swimming miracidia and surfacedwelling<br />
snail species were higher, and thus the lentic<br />
species were found to be infected maximally, compared<br />
to the bottom-dwelling species such as snails of<br />
Planorbidae, Viviparidae and Thiaridae family as they<br />
had little chances of contact with miracidia of various<br />
species. Running water (lotic habitat) diminishes the<br />
chance of interaction between miracidia and snails, and<br />
consequently minimizes the incidence of larval<br />
infection. Similar observations have been made <strong>for</strong><br />
cercarial infection in lotic waters (Radke et al., 1961;<br />
Babikar et al., 1984; Choubisa, 1986a,b). <strong>The</strong>se<br />
workers concluded that the infectivity was inversely<br />
proportional to the velocity of water flow but the<br />
diversity of different cercariae is much more dependent<br />
upon the member of different final hosts, which inhabit<br />
and frequently visit the water bodies as well as the<br />
density of miracidia and the population-size of snails<br />
species.<br />
<strong>The</strong> emergence of two types of cercariae from a single<br />
snail host is referred to as a case of double infection.<br />
Such cases were rarely observed in the present study.<br />
Such type of infections have also been observed by<br />
Mukherjee (1966), Jain (1970), Pandey and Agarwal<br />
(1978) and Choubisa (1986b). It is possible because of<br />
the identical host-specificity of two different digentic<br />
trematode parasites.<br />
Most of the cercariae emerge through out the year or in<br />
the all reasons with varying incidence of infection.<br />
However, the maximum incidence of infection was<br />
found in the late rainy or pre-winter season. It is a fact<br />
that rain-water is responsible <strong>for</strong> the aggregation of<br />
different types of miracidium larvae in the aquatic<br />
habitats. Secondly, the rain-water increases the snail<br />
population. Because almost all the fresh water snail<br />
species breed in the rainy season, most of the cercariae<br />
are released from their snail hosts in the late rainy<br />
season or pre-winter season. Simlar observations have<br />
also been reported by Mukherjee (1966), Mohandas<br />
(1974), and Pandey and Agarwal (1978). <strong>The</strong>se workers<br />
have reported two peaks of cercariael infection in a<br />
year, as has been observed in the present study.<br />
However, looking at the variability in the cercarial<br />
infection from place to place and year to year, it is<br />
proposed that the peaks of cercarial infection with a
Pathogenic trematode cercariae infecting freshwater snails in Rajasthan<br />
53<br />
Table I. Intermediate snail hosts and their trematode cercarial larvae<br />
Snail host species Habit/ Habitat Trematode cercarial larvae<br />
(A) Lymnaeidae:<br />
Amphi Echino Furco Gymno Mono Xiphi<br />
(i) Lymnaea acuminata f. patula Sd/Le - ++ - ++ - ++<br />
(ii) L. acuminata f. chlamys Sd/Le - ++ - ++ - ++<br />
(iii) L. acuminata f. typical Sd/Le - ++ - ++ - ++<br />
(iv) L. luteola f. austrails Sd/Le - ++ - ++ - ++<br />
(v) L.luteola f. typical Sd/Lo - ++ - ++ - ++<br />
(vi) L. luteola f. impure Sd/Lo - ++ - ++ - ++<br />
(vii) L. luteola f. rufescens Sd/Lo - ++ - ++ - ++<br />
(B)<br />
Planorbidae:<br />
(i) Indoplanorbis exustus Bd/Lo , Le ++ + ++ - - +<br />
(ii) Gyraulous convexiusculus Bd/Lo, Le ++ + ++ - - +<br />
(C) Viviparidae:<br />
(i) Vivipara bengalensis race gigantea Bd/Lo , Le - + ++ - - ++<br />
(ii) V. bengalensis race mandiensis Bd/Lo, Le - + ++ - - ++<br />
(D) Thiaridae:<br />
(i) Melania Striatella tuberculata Bd/Lo, Le - - ++ - ++ +<br />
(ii) Faunus ater Bd/Lo, Le - - ++ - ++ +<br />
(iii) M. (Plotia) scabra Bd/ Lo - - ++ - ++ -<br />
(iv) Thiara ( Tarebia) lineata Bd/ Lo - - ++ - ++ -<br />
++, commonly infected; +, rarely infected; -, non-infected; Bd, bottom-dweller; Le, lentic (stagnant water); Lo, lotic<br />
(running water); Sd, surface dweller.<br />
Amphi, amphistome; Echino, echinostome; Furco, furcocercous; Gymno, gymnocephalous; Mono, monostome; xiphi,<br />
xiphidiocercaria.<br />
Table II. Seasonal occurrence of trematode larvae (cercariae)<br />
Types of cercariae *Snail species Seasons<br />
Rainy Winter Spring Summer<br />
(Jul-Sept) (Oct-Jan) (Feb-Mar) (Apr-Jun)<br />
Amphistome Genera of Planorbidae ++ +++ + +<br />
Echinostome Genera of Lymnaeidae + +++ ++ +<br />
Furcocercous Genera of Viviparidae ++ +++ ++ +<br />
Gymnocephalous Genera of Lymnaeidae ++ +++ + +<br />
Monostome Genera of Thiaridae + +++ ++ +<br />
Xiphidio Genera of Lymnaeidae + +++ ++ +<br />
+++, > 90% cercariae infection; ++, 30-60% cerarial infection<br />
+, < 30% cercariae infection; -, infection is not found; * as in Table-I.
54<br />
Choubisa<br />
Table III. Relative behaviours of cercariae<br />
Types of cercariae<br />
Active<br />
Negative- Photo- Thigmotaxism<br />
Swimming geotaxism tropism<br />
Amphistome + + +++ -<br />
Echinostome + + ++ -<br />
Furcocercous +++ +++ +++ +++<br />
Gymnocephalous + + + -<br />
Monostome +++ +++ +++ ++<br />
Xiphidio+ ++ + -<br />
+++, More; ++, Moderate; +, less; -, no response .<br />
particular snail species are not stable but vary due to REFERENCES<br />
change in the physico-chemical factors of a particular Babiker A, Fenwick A and Amin MA. 1984 . Localization of<br />
niche (Choubisa , 1986a). It is interesting to note that Biomphalaria pfeifferi by Schistosoma mansoni miracidia in<br />
except furcocercous and xiphidio cercariae, others stagnant and running water under field conditions.<br />
showed stern host-specificity. Such observations have Hydrobiologia 110:235-240.<br />
rarely been observed and discussed (Choubisa 1986a, Cheng TC.1973. General <strong>Parasitology</strong> Academic Press, New<br />
1991). However, such findings are more useful <strong>for</strong> York and London.<br />
<strong>for</strong>ecasting the endemicity of a particular trematodiasis<br />
Choubisa SL. 1986a. <strong>The</strong> biology of certain larval trematods<br />
disease in a particular area.<br />
infecting fresh water snails of Lakes of Udaipur. Ph. D <strong>The</strong>sis,<br />
M. L. S. University, Udaipur.<br />
Strong phototactic behaviour among different cercariae<br />
is much more related to the presence of eye pigments on Choubisa SL. 1986b. Incidence of larval trematodes infection<br />
their body (furcocercous, monostome and amphistome and their seasonal variation in the fresh water of molluscs of<br />
cercariae). Active swimming of cercariae (furcocercous southern Rajasthan. Rec Zool Sur India 83:69-80.<br />
and monostome) is associated with the morphological Choubisa SL. 1991. Comparative study on cercarial behaviours<br />
adaptive characters such as small-sized main body, and their host specificity. Ind J of Parasit 15:125-128.<br />
prominent spinosed and muscularized bifurcated fail<br />
Choubisa SL. 1992. Molluscs as bio-indicators <strong>for</strong> the trophic<br />
(furcocercous), and ventral and dorsal fins on tail stages of lakes and lotic environments. Bull of Pur Appl Sci<br />
(monostome). However, furcocercous cercariae are 11:35-40.<br />
relatively more active swimmers as compared to<br />
Choubisa SL and Sharma PN. 1983. Seasonal variation of<br />
monostome, although both types of cercariae are active<br />
cercarial infection in snails of Fateh Sagar lake of Udaipur.<br />
swimmers. Additionally, such cercariae are strong Ind J Parasit 7:111-113.<br />
negative-geotaxic as they can move against the gravity.<br />
Erasmus DA. 1972. <strong>The</strong> Biology of Trematodes. Printed in Great<br />
<strong>The</strong> findings of the present study can be useful in the Britain at the University Press, Belfast.<br />
preparation of future health plans in tribal regions <strong>for</strong> Clarke AA.1979. Gastropods as indicators of trophic lake stages.<br />
the preservation of the health of tribals and their <strong>The</strong> Nautilus 94:138-142.<br />
domesticated animals, in understanding the biology of<br />
Harman WN.1974. Snails (Mollusca: Gastropoda). In: CW Hart<br />
trematode parasites, and to significantly add to the<br />
Jr. and S. L. H. Fuller (Eds.), Pollution Ecology of Fresh<br />
existing knowledge of parasitology.<br />
water Invertebrates (Academic Press, New York).<br />
ACKNOWLEGEMENTS<br />
Jain SP. 1970. Double infection of larval trematodes in molluscs.<br />
Agra Univ J Res Sci 21:47-48.<br />
I thank the University Grants Commission, New Delhi<br />
Mohandas A. 1974. Studies on the fresh water cercariae of Kerala<br />
<strong>for</strong> the financial assistance (F.No.4S-09/2004-<br />
I. Incidence of infection and seasonal variation. Folia Parasit<br />
2005(MRP/CRO)/203008). <strong>The</strong> author is grateful to 21:311-317.<br />
Prof. P. N. Sharma (Parasitologist) <strong>for</strong> his astute advice<br />
and valuable comments.<br />
Mukerjee RP.1966. Seasonal variations of cercarial infections in<br />
snails. J Zool Soc Ind 18:39-45.
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Mukerjee RP. 1980. Collection and study of larval trematods Schistosoma mansoni cercaria released under laboratory and<br />
(Platyhelminthes). Proc Workshop Tech Parasit Zool Sur Ind field conditions. Expl Parasit 11:323-331.<br />
23-26.<br />
Ray HC and Mukherjee I. 1963. Fauna of Rajasthan, India. Part 3<br />
Pandey KC and Agarwal N. 1978. Larval trematodes and Mollusca. Rec Zool Sur Ind 61:403-436.<br />
seasonal variations in snails of Kathauta Tal, Lucknow. Ind J<br />
Parasit 2:119-120.<br />
Tonapi GT. 1980. Fresh Water Animals of India. Ox<strong>for</strong>d and I. B.<br />
H. Publishing Company, New Delhi, India.<br />
Radke MG, Ritchie LS and Rowan WB. 1961. Effects of water<br />
velocities on worm burdens of animals exposed to
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 56-59<br />
Original paper<br />
J P D<br />
On Paralueheia guptai n. gen. & n. sp. (Acanthocephala:<br />
Plagiorhynchinae Meyer, 1931) from Stromateus sinensis<br />
A. M. Saxena and Rahul Gupta<br />
Department of Zoology, University of Lucknow, Lucknow.<br />
Received 24 March <strong>2008</strong>; revised 19 May <strong>2008</strong>; accepted 10 September <strong>2008</strong><br />
ABSTRACT. Paralueheia guptai n. gen. & n. sp. from the intestine of a marine fish Stromateus<br />
sinensis Day, from Deegha, West Bengal, India, is described. As compared with other genera of<br />
subfamily Plagiorhynchinae Meyer, 1931, the new genus was found to possess unique morphoanatomical<br />
characters: proboscis having 20 longitudinal rows of hooks with 17–18 hooks per row;<br />
testes pre-equatorial and tandem; two cement glands. Diagnostic characters of the new genus and a<br />
key of genera are provided.<br />
Keywords: Acanthocephala, Deegha, intestine, Paralueheia guptai, Stromateus sinensis Day.<br />
INTRODUCTION<br />
<strong>The</strong> Acanthocephala are endoparasites which lack a<br />
digestive tract in all stages of their development. <strong>The</strong><br />
characteristic feature of the phylum is an organ of<br />
attachment consisting of an invaginable proboscis,<br />
armed with rows of re-curved hooks (Hyman, 1951).<br />
Out of 800 known species, 229 (28.62%) of them have<br />
been reported from India (Alfred, 1998), including 203<br />
species native to <strong>Indian</strong> region (Bhattacharya, 1998).<br />
This indicates a very high percentage of prevalence.<br />
Fishes have been found to be more susceptible to<br />
infection of Acanthocephala (Milton and Franson,<br />
1999; Taraschewski, 2000). In the present paper, a new<br />
genera and species is described as Paralueheia guptai<br />
n. gen. & n. sp. (Fig. 1–4) from the intestine of a marine<br />
fish Stromateus sinensis Day (Fig. 5), recovered at<br />
Deegha, West Bengal, India.<br />
MATERIALS AND METHODS<br />
A large number of marine fishes were collected from the<br />
Corresponding author: Dr. A. M. Saxena, Department of<br />
Zoology, University of Lucknow, Lucknow-226 007, India.<br />
Email: anandmsaxena@rediffmail.com<br />
sea coast of Deegha, West Bengal, and dissected out and<br />
examined. During the examination, two male<br />
Acanthocephala specimens were recovered from the<br />
intestine of S. sinensis. <strong>The</strong> parasites were thoroughly<br />
washed and then kept in refrigerator till complete<br />
reversion of proboscis. Later, the parasites were<br />
flattened under slight pressure of cover glass and kept in<br />
A. F. A. fixative (50% alcohol, <strong>for</strong>maline and acetic acid<br />
in ratio of 100:6:2.5) <strong>for</strong> 24 h. After fixation, the<br />
parasites were stained in acetoalum carmine,<br />
differentiated in acid alcohol, dehydrated through<br />
ascending series of alcohol, cleared in xylol and then<br />
mounted in Canada balsam. Diagrams were made with<br />
the aid of Camera Lucida. All measurements are in<br />
millimeter.<br />
Type Description: Body is elongate, spinose,<br />
cylindrical and slightly broader at middle and narrow at<br />
extremities. Size is 4.2–10.6 mm long and 0.60–1 mm<br />
wide. Proboscis is sub-globular, short, 0.56–1.2 mm<br />
long and 0.38–0.66 mm wide, and having 20<br />
longitudinal rows of hooks with 1–18 hooks per row.<br />
Proboscis hooks are not uni<strong>for</strong>m. Hooks of anterior<br />
region are 0.060–0.062 mm long, of middle region
A n. gen. & n. sp. from Stromateus sinensis<br />
57<br />
Fig. 1-4. Paralueheia guptai n. gen. & n. sp.<br />
Fig. 1. Entire male (ventral view).<br />
Fig. 2. Male proboscis enlarged (ventral view).<br />
Fig. 3. Single row of proboscis hooks of male.<br />
Fig. 4. Proboscis hooks enlarged.<br />
0.052–0.056 mm long and of posterior region number, long, tubular, and 0.96–2.17 mm long and<br />
0.041–0.048 mm long. Neck is short and 0.18–1.06 mm 0.06–0.14 mm wide. Seminal vesicle is pyri<strong>for</strong>m, and<br />
long. Trunk is spinose at anterior region, cylindrical, 0.22–0.33 mm long and 0.07–0.25 mm wide. Bursa is<br />
and 3.46–7.9 mm long and 0.60–1 mm wide. Proboscis globular or pyri<strong>for</strong>m, without bursal rays, and<br />
receptacle is double walled and clavi<strong>for</strong>m, and 0.31–0.52 mm long and 0.16–0.34 mm wide. Genital<br />
1.05–3.15 mm long and 0.22–0.26 mm wide. Lemnisci pore is terminal.<br />
are equal, extending up to middle of anterior testis, and<br />
0.56–1.1 mm long. Testes are ovoid or elongate,<br />
DISCUSSION<br />
tandem, pre-equatorial, subequal, and apart from each <strong>The</strong> present <strong>for</strong>m of the parasite has been referred to<br />
other. Anterior testis is 0.14–0.21 mm long and subfamily Plagiorhynchinae Meyer, 1931 of the family<br />
0.08–0.16 mm wide, and at 1.96–2.89 mm from Plagiorhynchidae Golvan, 1960 in having numerous<br />
anterior end of the trunk. Posterior testis is 0.2–0.21 mm hooks on sub-globular proboscis; lemnisci slender,<br />
long and 0.09–0.17 mm wide, and at 2.11–3.10 mm large and without distinct central canal, and cement<br />
from anterior end of the trunk. Cement glands are two in
58 Saxena and Gupta<br />
Fig. 5. Stromateus sinensis Day<br />
Table I. Comparative chart of the genera of the subfamily Plagiorhynchinae Meyer, 1931<br />
Genus Plagiorhynch Lueheia Oligoterorhync Pseudogordiorhync Paralueheia new<br />
Structures us Luhe, 1911 Travassos, hus Monticelli, hus Golvan, 1957 genus<br />
1919 1914<br />
Body shape Plump, Fusi<strong>for</strong>m Elongate Elongate<br />
Elongate<br />
fusi<strong>for</strong>m<br />
Proboscis Cylindrical or Subglobular, Ovoid, 14 Tunicated, 28-34 Subglobular,<br />
and subcylindrical, 22-30 longitudinal longitudinal rows 20 longitudinal<br />
proboscis 18-22 longitudinal rows with with 12-13 hooks rows with 17 to<br />
hooks longitudinal rows with 6-7 hooks per row 18 hooks per row<br />
rows with 8-10 hooks per row<br />
10-12 hooks per row<br />
per row<br />
Proboscis Subcylindrical Clavi<strong>for</strong>m Subcylindrical Elongate Clavi<strong>for</strong>m<br />
receptacle<br />
Lemnisci Unequal, Unequal, Equal, Equal, Equal,<br />
branched, unbranched, unbranched, unbranched, unbranched,<br />
slender, 2 tubular, 4-6 tubular, 2 flattened, 2 tubular,2<br />
Testes Preequatorial Preequatorial Postequatorial, Preequatorial, Preequatorial,<br />
continuous tandem continuous<br />
tandem<br />
Cement 4, 6 or 8 in 4 in number 4 in number 3 in number 2 in number<br />
glands number<br />
glands tubular. Subfamily Plagiorhynchinae comprises proboscis hooks with 17–18 hooks per row. <strong>The</strong>re are<br />
of the following genera: Plagiorhynchus Luhe, 1911; 18–22 longitudinal rows of proboscis hooks with 8–10<br />
Pseudogordiorhynchus Golvan, 1957; Lueheia hooks per row in Plagiorhynchus, 22–30 longitudinal<br />
Travassos, 1919 and Oligoterorhynchus Monticelli, rows with 8–10 hooks per row in Lueheia, 14<br />
1914 (Yamaguti, 1961). <strong>The</strong> present <strong>for</strong>m differs from longitudinal rows with 6–7 hooks per row in<br />
all these genera of subfamily Plagiorhynchinae in Oligoterorhynchus, and 28–34 longitudinal rows with<br />
having two cement glands, and 20 longitudinal rows of 12–13 hooks per row in Pseudogordiorhynchus. <strong>The</strong>re
A n. gen. & n. sp. from Stromateus sinensis<br />
59<br />
are 4–8 cement glands in Plagiorhynchus, three in with 8–10 hooks per row…… Lueheia Travassos,<br />
Pseudogordiorhynchus, four each in Lueheia and 1919<br />
Oligoterorhynchus.<br />
Proboscis having 28–34 longitudinal rows of hooks<br />
<strong>The</strong> present <strong>for</strong>m can be further distinguished from with 12–13 hooks per row …<br />
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<br />
Pseudogordiorhynchus in having sub-globular<br />
proboscis; from Lueheia in having lemnisci two and<br />
ACKNOWLEDGMENT<br />
equal, and testes tandem; from Oligoterorhynchus in Authors are thankful to Professor Nirupama Agarwal,<br />
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,<br />
Pseudogordiorhynchus in having testes tandem (Table Lucknow <strong>for</strong> her constant encouragement and guidance<br />
I). Accordingly, it is not possible to assign the present in this work.<br />
<strong>for</strong>m under any genus of the subfamily<br />
Plagiorhynchinae. <strong>The</strong>re<strong>for</strong>e, it deserves the status of a REFERENCES<br />
new genus Paralueheia, with type species P. guptai n. Alfred JRB. 1998. Faunal Diversity in India-An Overview. In:<br />
gen. & n. sp., proposed <strong>for</strong> its reception.<br />
Faunal Diversity in India. Volume I-VIII, ENVIS Centre,<br />
Zoological Survey of India, Calcutta, India, pp. 1-495.<br />
<strong>The</strong> new species is named in the honour of late<br />
Bhattacharya SB. 1998. Faunal diversity in India. In:<br />
professor S. P. Gupta, D. Sc., Department of Zoology,<br />
Acanthocephala. Envis Centre, Zoological Survey of India,<br />
University of Lucknow, Lucknow. Calcutta. India, pp. 93-98.<br />
Paralueheia n. gen.<br />
Golvan YJ. 1957. Acanthocephales d'oiseaux. Sixieme note:<br />
Deux especes nouvelles parasites d'oiseaux d'Afrique<br />
Generic diagnosis: Family-Plagiorhynchidae; occidentale Française: Gordiorhynchus (Gordiorhynchus)<br />
subfamily-Plagiorhynchinae; body elongate, spinose gendrei n. sp. et Pseudogordiorhynchus antonmeyeri n. g. n.<br />
and cylindrical. Proboscis subglobular, having 20 sp. Annals de <strong>Parasitology</strong> Hum Comp 32:83-97.<br />
longitudinal rows of hooks with 17–18 hooks per row, Golvan YJ. 1960. Le Phylum des Acanthocephala (3. note). La<br />
hooks large at apex and small at the base of proboscis. classe de Palaeacanthocephala Meyer, 1931. Annals de<br />
Proboscis receptacle double-walled and clavi<strong>for</strong>m. <strong>Parasitology</strong> Hum Comp 35:138-165.<br />
Lemnisci two, equal, tubular, and extending up to<br />
Hyman LH. 1951. Acanthocephala, Aschelminthes and<br />
middle of anterior testis. Testes ovoid or elongate, Entoprocta. In: <strong>The</strong> Invertebrates, Volume-3, First ed.,<br />
subequal, preequatorial, and tandem. Cement glands McGraw-Hill Publishers, USA. pp. 1-49.<br />
two, long and tubular. Seminal vesicle pyri<strong>for</strong>m. Bursa<br />
Luhe M. 1911. Acanthocephalen, Brauer Susswasserfauna<br />
globular or pyri<strong>for</strong>m without bursal rays. Genital pore<br />
Deutschlands. Heft, pp. 16.<br />
terminal. Parasite in marine fishes.<br />
Meyer A. 1931. Die Acanthocephalen der arktischen Gebietes.<br />
Type species: Paralueheia guptai n. gen. & n. sp. Fauna Arctica 6:9-20.<br />
Key to the genera of the subfamily Plagiorhynchinae<br />
Meyer, 1931<br />
Milton F and Franson JC. 1999. Acanthocephaliasis. In: Field<br />
Mannual of Wildlife Disease, US Department of Interior, US<br />
Geology Survey, US, pp. 241-243.<br />
1. Cement glands 2…… Paralueheia n. gen. & n. sp. Taraschewski H. 2000. Host-parasite Interactions in<br />
Cement glands more than 2… 2<br />
Acanthocephala: A Morphological Approach. Advances in<br />
2. Testes preequatorial …… 3<br />
<strong>Parasitology</strong> 46:1-179. \<br />
Travassos, L. 1919. In<strong>for</strong>macoes sobre o material<br />
Testes postequatorial …… Oligoterorhynchus helminthologico colleccionado na Ilha da Trinidade em,<br />
Monticelli, 1914 1916. Arquivos do Museu Nacional Rio de Janeiro 22:159-<br />
(Yamaguti, 1963)<br />
167.<br />
Yamaguti S. 1963. Oligoterorhynchus Monticelli, 1914. In:<br />
3. Lemnisci branched …… Plagiorhynchus Systema Helminthum-Acanthocephala. Volume-5, John<br />
Luhe, 1911 Wiley & Sons, New York, pp 91.<br />
Lemnisci unbranched …… 4<br />
4. Proboscis having 22–30 longitudinal rows of hooks
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 60-63<br />
Original paper<br />
J P D<br />
Some epidemiological aspects of intestinal cestodes of<br />
sheep in a temperate climate<br />
K. A. Tariq, M. Z. Chishti and F. Ahmad<br />
Post Graduate Department of Zoology, <strong>The</strong> University of Kashmir, Srinagar.<br />
Received 5 <strong>June</strong> <strong>2008</strong>; accepted 10 September <strong>2008</strong><br />
ABSTRACT. <strong>The</strong> seasonal prevalence of intestinal cestode parasites in association with sex, breed<br />
and age of sheep, from February 2005 to January 2007 in Kashmir valley was studied.<br />
Gastrointestinal tract examinations of slaughtered sheep were conducted monthly to monitor the<br />
seasonal prevalence of cestodes. During the period of two years, a total of 498 sheep were examined<br />
and 178 of them were found positive <strong>for</strong> infection. <strong>The</strong> overall prevalence of the parasites of<br />
st<br />
nd<br />
anoplocephalidae family was 35.7% (1 year: 34.05% and 2 year: 37.2%). <strong>The</strong> parasites in<br />
decreasing order of prevalence were Moniezia expansa (6%), Avitellina centripunctata (3.7%), Stilesia<br />
globipunctata (2.8%) and Thysaneizia giardi (2.2%). Highest infection was recorded in autumn<br />
among the males of nomad breed (p > 0.05) vis-à-vis those of adult females of local breed. In<br />
conclusion, we recorded quite low prevalence of anoplocephalidae in sheep of Kashmir valley<br />
although season, age, sex and breed were the factors that influenced cestode infection in sheep.<br />
Keywords: age, anoplocephalidae, breed, epidemiology, prevalence, sex, sheep<br />
INTRODUCTION<br />
Production of sheep is an attractive profession of<br />
farmers in Kashmir valley, India, due to low capital<br />
input and the ability of sheep to thrive on native<br />
meadows and agricultural fields. <strong>The</strong>re being limited<br />
scope <strong>for</strong> industrialization in the valley, varied<br />
activities of agriculture including sheep-rearing has<br />
become a major source of income. Almost every rural<br />
household rears sheep, which caters to their daily needs<br />
of milk, meat, wool, leather and valuable organic<br />
manure. Sheep are subjected to infection of diverse<br />
types of helminth parasites particularly the soil- and<br />
grass-transmitted species because of their grazing<br />
habits. Anoplocephalids (intestinal cestodes) <strong>for</strong>m an<br />
Corresponding author: Mr. K. A. Tariq, <strong>Parasitology</strong> Research<br />
Laboratory, Post Graduate Department of Zoology, <strong>The</strong><br />
University of Kashmir, Srinagar-190 006, India.<br />
E-mail: drkatariq@yahoo.co.in<br />
important group of intestinal helminth fauna of sheep.<br />
<strong>The</strong> life cycles of intestinal cestodes in sheep are<br />
indirect and herbage mites (Galumna, Oribatula,<br />
Peloribates, Scheloribates, etc.) of the family<br />
Oribatidae and psocids act as their intermediate hosts.<br />
During feeding, the mites and psocids accidentally<br />
ingest eggs of the tapeworms present in the faecal<br />
matter and on the grass, and the larval stage called a<br />
cysticercoid develops in them. Sheep become infected<br />
by ingesting herbage containing mites carrying the<br />
infective stage of the parasites (Soulsby, 1982).<br />
Planning of control strategies of helminthosis requires<br />
knowledge of the epidemiological factors that<br />
influence the risk of helminth infections. To-date no<br />
detailed epidemiological work has been carried out on<br />
intestinal cestodes of sheep in Kashmir valley, hence we<br />
took up the present study. <strong>The</strong> objective of this study<br />
was to investigate the epidemiological aspects of
Intestinal cestodes of sheep<br />
61<br />
intestinal cestode parasites of sheep reared under<br />
traditional husbandry conditions in Kashmir valley.<br />
MATERIALS AND METHODS<br />
This study was carried out in sheep slaughtered in<br />
various abattoirs of the Kashmir valley from February<br />
st<br />
st<br />
1 2005 to January 31 2007. A total of 498<br />
gastrointestinal tracts of sheep were examined during<br />
this period. All the sheep examined belonged to<br />
Corriedale, Bhakarwal, Kashmir Merino and Kashmiri<br />
breed. All the animals belonged to both sexes and their<br />
age ranged from < one year to > four years. Monthly<br />
collection of the samples was carried out to ensure the<br />
seasonality of infection. <strong>The</strong> gastrointestinal tracts<br />
carefully labeled with animal identification, breed, sex,<br />
dental age and month of collection, were collected fresh<br />
in normal saline (0.9%), and analyzed in the laboratory.<br />
Abomasum, small and large intestines were analyzed<br />
separately using standard procedures and the recovered<br />
parasites were identified on the basis of morphological<br />
descriptions of Soulsby (1982).<br />
RESULTS<br />
<strong>The</strong> overall prevalence of anoplocephalidae in sheep<br />
was 35.7% (year 1: 34.05% and year 2: 37.2%). <strong>The</strong><br />
parasites in decreasing order of prevalence were<br />
Moniezia expansa (6.0%), Avitellina centripunctata<br />
(3.7), Stilesia globipunctata (2.8) and Thysaneizia<br />
giardi (2.2%). <strong>The</strong> data pooled <strong>for</strong> seasonal estimation<br />
of cestode infection revealed maximum cestode<br />
infection (Year 1: 41.6%; Year 2: 46.8%) in autumn and<br />
lowest (Year 1:19.2%; Year 2: 22.4%) in winter (p ><br />
0.05 (year 1: p = 0.254; year 2: p = 0.204). <strong>The</strong> seasonal<br />
prevalence of infection has been shown in Table I.<br />
Influence of sex and breed and age on the prevalence<br />
of infection<br />
<strong>The</strong> present study revealed that sex, breed and age of the<br />
host influence the prevalence of intestinal cestode<br />
infection in sheep (Table II). Comparatively, males<br />
were more infected than females. <strong>The</strong> breed wise<br />
investigation of infection revealed low prevalence in<br />
Table I. Seasonal prevalence of intestinal cestode<br />
parasites in sheep over a period of two years<br />
Season Year Examined Infected (%)<br />
Spring Year 1 60 19 (31.6)<br />
Year 2 72 22 (30.5)<br />
Summer Year 1 60 24 (40.0)<br />
Year 2 72 29 (40.2)<br />
Autumn Year 1 60 25 (41.6)<br />
Year 2 64 30 (46.8)<br />
Winter Year 1 52 10 (19.2)<br />
Year 2 58 13 (22.4)<br />
Mean ± SE Year 1 58±2.0 19.5±3.42; p = 0.254<br />
Year 2 66.5±3.4 23.5±3.92; p = 0.204<br />
local Kashmiri breed. <strong>The</strong> Bhakarwal and Corriedale<br />
breed were more infected. <strong>The</strong> cross bred Kashmir<br />
Merino was found to harbour intermediate level of<br />
infection. However, the observations were statistically<br />
insignificant (p > 0.05). <strong>The</strong> age wise observations<br />
revealed highest prevalence in lower age groups.<br />
Generally, less than one year old animals were more<br />
infected and with the increase in age, the infection level<br />
decreased.<br />
DISCUSSION<br />
Percentages with their respective means ± SE (standard<br />
error of the mean) were employed to calculate the<br />
prevalence of anoplocephalidae infection. Chi-square<br />
test was employed to measure association between the<br />
parasitism and season, age, sex and breed, and the<br />
values were considered significant at p < 0.05. <strong>The</strong> data<br />
was analyzed using statistical packages MINITAB-<br />
13.20 <strong>for</strong> windows.<br />
<strong>The</strong> observations of low prevalence of<br />
anoplocephalidae seem to be in relation to the agro<br />
climatic conditions of the valley. Similar parasites with<br />
different prevalence's have also been reported in other<br />
parts of the country and world under similar or different<br />
climates. Kiran et al. (2005) found M. expansa as the<br />
most prevalent helminth in sheep in Dehradun. Munib<br />
et al. (2004) reported the overall prevalence of 28% of<br />
M. expansa, M. benedeni and A. centripunctata.<br />
Muraleedharan (2005) reported 1.65% prevalence of<br />
Moneizia spp. in sheep. In Turkey, Aydenizoz and<br />
Yildiz (2003) also reported low prevalence of M.<br />
expansa (3.98%) and A. centripunctata (0.86%) in<br />
sheep. M. expansa, M. benedeni, T. giardi and A.<br />
centripunctata were also recovered from sheep in<br />
Moldova (Zgardan, 2002).<br />
<strong>The</strong> present study revealed definite seasonality of<br />
infection and in line with this seasonal trend are the<br />
observations of various authors (Soulsby, 1982;<br />
Courteny et al., 1985). In Turkey, highest infection of<br />
M. expansa, A. centripunctata, Thysaneizia ovilla in<br />
sheep was observed in July (9.89%) and the lowest in<br />
September (1.32%; Aydenizoz and Yildiz, 2003). <strong>The</strong><br />
over-wintering of mites on pastures and subsequent
62 Tariq et al.<br />
Table II. Influence of sex, breed and age on infection over a period of two years<br />
Factor Level Year 1 Year 2<br />
Examined Infected (%) p-value Examined Infected (%) p-value<br />
Sex Male 112 39(34.8) 0.792 140 50(35.7) 0.926<br />
Female 120 39(32.5) 126 44(34.9)<br />
Mean ± SE 116±4.0 39±0.0 133±7.0 47±3.0<br />
Breed Corriedale 64 23(35.9) 69 27(39.1)<br />
Kashmiri 50 16(32.0) 59 17(28.8)<br />
Bhakarwal 67 24(35.8) 0.944 76 31(40.7) 0.682<br />
Kashmir Merino 51 15(29.4) 62 19(30.6)<br />
Mean ± SE 58±4.37 19.5±2.32 66.5±3.79 23.5±3.30<br />
Age 4 35 10(28.5) 41 11(26.8 )<br />
Mean ± SE 46.4±3.77 15.6±2.15 53.2±5.42 18.8±3.91<br />
infection of animals in the following grazing months et al., 2004; Wildeus and Zajac, 2005) where some<br />
may account <strong>for</strong> high prevalence in autumn season. As breeds were found to be more susceptible to helminth<br />
the whole Kashmir valley remains snow covered in infection as compared to the others.<br />
winter season, there<strong>for</strong>e, no fresh infection with these<br />
anoplocephalidae occurs in winter. <strong>The</strong> infection to<br />
<strong>The</strong> higher infection in lower age groups may be due to<br />
sheep might be occurring somewhere in spring season<br />
the low resistance in young animals. It is a well<br />
and early summer every year when the animals are sent<br />
established fact that lambs are more prone to cestode<br />
<strong>for</strong> grazing in the pastures and agricultural lands. After<br />
infection particularly in case of M. expansa and become<br />
completing the required prepatent period in sheep, the<br />
infected very early in life (Soulsby, 1982). A high<br />
infection starts appearing in highest numbers in the<br />
incidence of enterotoxaemia has been associated with<br />
autumn season of respective years as was significantly<br />
Moneizia infections in lambs in USSR (Vibe, 1976).<br />
observed during the two years of the present study.<br />
<strong>The</strong> low level of parasitism reported in adult animals is<br />
due to the development of significant immunity.<br />
<strong>The</strong> influence of sex on the susceptibility of animals to Similar findings are available from other studies also.<br />
infections could be attributed to genetic predisposition. For example, in different age groups of sheep, young<br />
Our results are in agreement with Gorski et al. (2004) animals were more prone to infection with intestinal<br />
who reported that males were reported to be more cestodes than adult animals (Rauf et al., 2005).<br />
infected. However, many studies have indicated that<br />
females were more infected than males (Patel et al.,<br />
In conclusion, the present endeavour reported quite low<br />
2001; Lateef et al., 2005). <strong>The</strong>re<strong>for</strong>e, sex of the animals<br />
prevalence of anoplocephalidae in sheep under<br />
cannot be regarded as a significant factor <strong>for</strong> cestode<br />
temperate agro climatic conditions of Kashmir valley<br />
infection; however, local management conditions can<br />
and we found season, age, sex and breed as important<br />
play a significant role in the occurrence of cestode<br />
factors that influenced the risk of cestode infection in<br />
infection in sheep despite the vice versa gender<br />
sheep.<br />
differences reported in several studies.<br />
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Journal of Agriculture and Biology 6:1128-1131.<br />
Yadav AK and Tandon V. 1989. Gastrointestinal nematode<br />
infections of goats in a subtropical and humid zone of India.<br />
Veterinary <strong>Parasitology</strong> 33:135-142.
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 64-67<br />
Short communication<br />
J P D<br />
Effect of the control of one-host cattle tick Boophilus<br />
microplus on the growth and haematological parameters<br />
of calves<br />
S. Vatsya, R. R. Kumar, C. L. Yadav and R. Garg<br />
Department of Veterinary <strong>Parasitology</strong>, G. B. Pant University of Agriculture and Technology, Pantnagar.<br />
Received 8 <strong>June</strong> 2007; revised 8 August 2007; re-revised 1 October 2007; accepted 3 January <strong>2008</strong><br />
ABSTRACT. A study was undertaken to understand the effect of the control of Boophilus microplus<br />
ticks on live-weight gain and various haematological parameters in cattle calves. Following<br />
application of Tickout (cypermethrin) @ 1 ml/liter, as spray wash, 94.68% control was attained, 3<br />
st nd rd th th th th<br />
days post-treatment. At the end of 1 , 2 , 3 , 4 , 5 , 6 and 8 week post-treatment (WPT), calves<br />
showed a percent weight gain of 7.77, 13.54, 25.42, 41.29, 62.42, 80.10 and 100.54, respectively.<br />
st nd rd th th th th<br />
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,<br />
35.12, 49.84, 67.76 and 87.80, respectively, was recorded in the untreated control calves. Tick<br />
infestation caused anaemia in calves. Acaricide treatment resulted in the elevation of haematological<br />
parameters, which reflected an ef<strong>for</strong>t on the part of calves to return to normal as compared to those in<br />
control group in which persistence of anaemia was evident.<br />
Keywords: Boophilus microplus, control, haematological parameters, weight gain<br />
Ticks, especially Boophilus microplus, are<br />
economically important pests of cattle in a tropical<br />
country like India. Despite the fact that animals remain<br />
infested with ticks throughout the year and have heavy<br />
tick burden during peak tick-season resulting in great<br />
production loss, it is treated as a casual entity. Though<br />
very few studies have been carried out regarding the<br />
impact of tick infestation on live-weight gain and<br />
effects on haematological parameters in cattle in<br />
different parts of the world (Johnnston et al., 1981;<br />
Williams and Buckner, 1977), such reports were<br />
difficult to be fished out from India. For this reason<br />
only, the present study was undertaken and the results<br />
are reported herein.<br />
Corresponding author: Dr. C. L. Yadav, Department of<br />
Veterinary <strong>Parasitology</strong>, G. B. Pant University of Agriculture<br />
and Technology, Pantnagar-263 145, India.<br />
Twenty two crossbred male calves (Jersey x Sahiwal;<br />
28-31 day old, weighing 22–32 kg), naturally-infested<br />
with B. microplus and maintained at Instructional Dairy<br />
Farm, Pantnagar, were used in this study. <strong>The</strong> animals<br />
were kept in yards made of concrete floors, and were<br />
given standard feed. <strong>The</strong> calves were randomly divided<br />
into two groups of eleven animals each. <strong>The</strong> animals of<br />
group-I (G-I) were treated with Tickout (cypermethrin;<br />
Alembic Veterinary Division, Vadodara) @ 1 ml/litre as<br />
spray wash and their shed @ 2ml/litre. <strong>The</strong> animals of<br />
group-II (G-II) were left untreated but kept separately<br />
in a different shed. Side counts of ticks were made<br />
(Holdsworth, 2006) be<strong>for</strong>e treatment, and on 3, 7 and<br />
thereafter at 15 days interval, post treatment. <strong>The</strong><br />
animals were weighed and their blood was collected <strong>for</strong><br />
haemoglobin (Hb), packed cell volume (PCV), total<br />
erythrocyte count (TEC), MCV, MCH and MCHC as<br />
st<br />
described by Jain (1993) at the start of the trial and 1 ,
Effect of the control of Boophilus microplus<br />
65<br />
Table I. Haematological alterations (Mean± SE) following treatment of cattle naturally infested with B. microplus<br />
Weeks post treatment<br />
Parameter 0 I II III IV V VI VII<br />
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<br />
(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<br />
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<br />
(%) 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<br />
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<br />
12<br />
(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<br />
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<br />
(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<br />
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<br />
(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<br />
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<br />
(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<br />
Vatsya et al.<br />
nd rd th th th th<br />
2 , 3 , 4 , 5 , 6 and 8 week after treatment (WPT). Tick infestation affects appetite, digestion and<br />
metabolism of animals, possibly by the secretion a<br />
Following application of cypermethrin, 94.68% control<br />
hepatotoxic compound (Jonsson, 2006). Lower weight<br />
was attained 3 days post-treatment (DPT). <strong>The</strong> animals<br />
gain has also been reported by Okello-Onen et al.<br />
became free of tick infestation 7 DPT, and their coat<br />
(2003) in un-dipped tick infested cattle. Sutherst et al.<br />
became shiny. In the untreated group, the number of<br />
(1983) observed that cattle heavily infested with ticks<br />
st<br />
ticks increased from 38.37±2.63 (1 week) to 102±3.15<br />
gained weight rapidly after removal of tick burden, as<br />
th<br />
at the end of trial (8 week). <strong>The</strong>se animals appeared compared to the un-infested animals. <strong>The</strong>se researchers<br />
weak, dull with rough coats.<br />
calculated the mean value <strong>for</strong> the effect of damage in<br />
th th<br />
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<br />
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<br />
nymphal stages of ticks in G-I. <strong>The</strong>re<strong>for</strong>e, all the 1.18±0.21g/engorging tick. Scholtz et al. (1991)<br />
infested animals in both the groups were treated with estimated the effect of individual engorged tick<br />
Tickout @ 1 ml/litre, at the end of experiment.<br />
in Here<strong>for</strong>d, Bonsmara and Nguni breeds of cattle to be<br />
In the treated group, the mean weight increased from 8.9, 8 and 8.6 g, respectively. Nguni breed carried least<br />
th<br />
27.54±0.84 kg to 55.23±0.72 kg by 8 WPT, with a<br />
number of B. decoloratus (5.30) and suffered from least<br />
percent weight gain of 7.77, 13.54, 25.42, 41.29, 62.42,<br />
reduction in weight gain. Williams et al. (1977), while<br />
80.10 and 100.54, respectively, at the end of<br />
studying the effects of Gulf Coast tick on weights of dry<br />
st nd rd th th th th<br />
1 ,2 ,3 ,4 , 5 ,6 and 8 WPT (Fig. 1). A major<br />
lot Here<strong>for</strong>d steers observed that, compared to control<br />
difference in the weight of animals was observed at the<br />
animals, high- and low-infested animals were 24 kg and<br />
rd<br />
14 Kg lighter, respectively. <strong>The</strong> growth rates of cattle<br />
end of 3 WPT. In the G-II animals, the mean weight<br />
were far better, besides reduction in the incidence of<br />
increased from 27.45±0.77 kg to 51.55±0.65 kg. At the<br />
st nd rd th th th th<br />
tick-born diseases, when strategic tick control measures<br />
end of 1 ,2 ,3 ,4 ,5 , 6 and 8 week, the percent<br />
are adopted as compared to no tick control or control<br />
weight gain were recorded to be 3.97, 12.60, 23.68,<br />
coupled with pasture spelling (Johnston et al., 1981).<br />
35.12, 49.84, 67.76 and 87.80, respectively.<br />
35<br />
Mean live weight gain (kg)<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
-5<br />
1 2 3 4 5 6 7<br />
Treated group<br />
Control group<br />
Weeks post treatment<br />
Fig. 1. Live weight gain in cattle calves infested with Boophilus microplus following acaricide application.
Effect of the control of Boophilus microplus<br />
67<br />
<strong>The</strong> results of the haematological studies are presented<br />
in Table-I. Following acaricide application, the<br />
haematological values viz. Hb, PCV, TEC were found<br />
elevated, which reflected an ef<strong>for</strong>t on the part of the<br />
animal to return to normal. Persistence of anaemia in<br />
the tick infested animal could be the result of blood-loss<br />
due to blood sucking activity of ticks. Ram et al. (2004)<br />
recorded mortality of 19 calves in a herd of 36 crossbred<br />
B. microplus-infested calves in the age group of<br />
6–18 months. <strong>The</strong> carcass of calves revealed severe<br />
anaemia and emaciation. <strong>The</strong> tick-infested surviving<br />
animals were extremely weak, anaemic, and anorectic<br />
and failed to gain weight.<br />
Association <strong>for</strong> the Advancement of Veterinary <strong>Parasitology</strong><br />
(WAAVP) guidelines <strong>for</strong> evaluation the efficacy of acaricides<br />
against ticks (Ixodidae) on ruminants. Vet Parasitol 136:29-<br />
43.<br />
Jain NC. 1993. Essentials of Veterinary Haematology. Lea and<br />
Febiger, Philadelphia, pp 417.<br />
Johnston LAY, Haydock KP, Leatch G. 1981. <strong>The</strong> effects of two<br />
systems of cattle tick (Boophilus microplus) control on tick<br />
populations, transmission of Babesia spp. and Anaplasma<br />
spp. on production of Brahman crossbred cattle in the dry<br />
tropics. Aust J Exp Agric Anim Husbandry 21:256-267.<br />
Jonsson NN. 2006. <strong>The</strong> productivity effects of cattle tick<br />
(Boophilus microplus) infestation on cattle with<br />
particular reference to Bos indicus cattle and their crosses. Vet<br />
Parasitol 137:1-10.<br />
<strong>The</strong> results of the present study suggest that ticks affect<br />
weight gain as well as various haematological Okello-Onen J, Tukahira EM, Perry BD, Rowlands GJ, Nagada<br />
parameters in cattle. <strong>The</strong>se observations assume SN, Musisi G, Bode E, Heinonen R, Mwayi W and Opuda-<br />
Asibo J. 2003. <strong>The</strong> impact of tick control on the productivity<br />
significance when production per<strong>for</strong>mance of animals<br />
of indigenous cattle under ranch conditions in Uganda. Trop<br />
is affected due to reduced weight gains, and is reflected<br />
Anim Hlth Prod 35:237-247.<br />
while taking decisions in adopting control measures in<br />
Ram H, Yadav CL, Banerjee PS and Kumar V. 2004. Tick<br />
the event of a tick outbreak. Thus there is a need <strong>for</strong><br />
associated mortality in crossbred cattle calves. <strong>Indian</strong> Vet J<br />
strategic control of tick infestation in animals so that the 81:1203-1205.<br />
productivity of animals remains unaffected.<br />
Scholtz MM, Spickett AM, Lombard PE, Eusulin CB. 1991. <strong>The</strong><br />
ACKNOWLEDGEMENTS<br />
effect of tick infestation on the productivity of cows of three<br />
breeds of cattle. Onderstepoort J Vet Res 58:71-74.<br />
<strong>The</strong> authors are thankful to the Dean, College of<br />
Sutherst RW, Maywald GF, Kerr JD, Stegeman DA. 1983. <strong>The</strong><br />
Veterinary and Animal Sciences, G. B. Pant University effect of cattle tick (Boophilus microplus) on the growth of<br />
of Agriculture and Technology, Pantnagar, and Joint Bos indicus X B. taurus steers. Aust J Agric Res 34:317-327.<br />
Director, Instructional Dairy Farm, Nagla, Pantnagar,<br />
Williams RE, Hair JA and Buckner RG. 1977. Effects of Gulf<br />
<strong>for</strong> their cooperation and help during the study period. Coast tick on blood composition and weights of dry lot<br />
Here<strong>for</strong>d steers. J Eco Entomol 70:229-233.<br />
REFERENCES<br />
Holdsworth PA, Kemp D, Green P, Peter RJ, Bruin CDe, Jonsson<br />
NN, Letonja T, Rehbein S and Vercruysse J. 2006. World
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 68-73<br />
Short communication<br />
J P D<br />
Mode of nutrition in pathogenic trematode larvae (redia<br />
and cercaria) which infect hepatopancreas of fresh water<br />
snails (Mollusca: Gastropoda)<br />
S. L. Choubisa<br />
Department of Zoology, Government Post Graduate College, Sirohi.<br />
Received 19 November 2007; accepted 2 September <strong>2008</strong><br />
ABSTRACT. Histochemical procedures were employed to demonstrate various hydrolytic enzymes<br />
in the pathogenic trematode larvae, rediae and cercariae in relation to the nutrition or digestion of the<br />
food. Tegument of both rediae and cercariae were found to be endowed with acid phosphatase, β-<br />
glucuronidase, esterase and lipase. <strong>The</strong>se hydrolases were localized in the gut of rediae, but caecal gut<br />
of cercariae of Echinostoma tewarii and Cercaria udaipuriensis contained only acid phosphatase with<br />
low activity. Comparison between the nutrition of rediae and cercariae in the present study revealed<br />
the <strong>for</strong>mer to be nutritionally advanced over the latter. Redia has been found to use both the routes to<br />
obtain nutrition i.e., tegument and caecal gut, whereas cercariae use tegument as evidenced by<br />
histochemistry of tegument. Localization of these hydrolases in other parts or organ systems of these<br />
larvae has also been discussed.<br />
Keywords: cercaria, hydrolases, nutrition, redia, trematode larvae<br />
Although, a large number of histochemical tests have<br />
been conducted in various larval <strong>for</strong>ms of digenetic<br />
trematodes by previous investigators (Fripp, 1966;<br />
Cheng and Yee, 1968; Clark, 1968; Porter and Hall,<br />
1970a,b; Choubisa, 1989), no systematic studies have<br />
been conducted on the enzymes involved in the<br />
digestion in these larvae. Sporocyst is devoid of any<br />
kind of digestive tract and, there<strong>for</strong>e, nutrients are<br />
absorbed through general body surface. Only rediae<br />
and cercaria are endowed with a simple digestive tract.<br />
Nevertheless, previous studies on nutrition of cercariae<br />
have clearly revealed them to be non-feeding and their<br />
caecae mostly devoid of digestive enzymes. Rediae of<br />
most of the trematodes are provided with oesophageal<br />
gland and long simple gut, and have received much<br />
attention <strong>for</strong> the presence of various enzymes involved<br />
Corresponding author: Dr. S. L. Choubisa, 205, Chanakyapuri,<br />
Sector 4, Hiran Magri, Udaipur-313 002, India.<br />
E-mail : choubisasl@yahoo.com<br />
in nutrition (Cheng, 1964; Probert, 1966; Erasmus,<br />
1972). <strong>The</strong> aim of the present study is to<br />
histochemically localize the presence of certain<br />
hydrolytic enzymes in some species of cercariae and<br />
rediae, hitherto uninvestigated.<br />
Two different unreleased cercariae, Echinostoma<br />
tewarii. (Choubisa and Sharma, 1985) and<br />
furcocercous cercariae, Cercaria udaipuriensis<br />
(Sharma and Choubisa, 1983) were collected by<br />
breaking the shell of fresh water snails Indoplanorbis<br />
exustus and Vivipara bengalensis, respectively. <strong>The</strong><br />
<strong>for</strong>mer species is released from birth-pore of rediae,<br />
whereas the letter directly from sporocysts. <strong>The</strong> size of<br />
C. udaipuriensis is larger than that of E. tewarii, and<br />
bears prominent oral sucker, pharynx, oesophagus,<br />
broad and crenated intestinal caeca, and are active<br />
swimmers. However, E. tewarii possesses these parts of<br />
digestive system but caeca was narrow and<br />
oesophagous surrounded by many penetration glands.
Mode of nutrition in pathogenic trematode larvae<br />
69<br />
Moreover, this species of cercariae has prominent<br />
distribution of many cystogenous glands over the body.<br />
<strong>The</strong> gut of rediae of E. tewarii contains brownish<br />
yellow macrogranules and extends up to three-fourth of<br />
the body.<br />
Various histochemical methods <strong>for</strong> the localization of<br />
certain enzymes such as lipase, acid phosphatase, -<br />
glucuronidase and simple esterase in these trematode<br />
larvae were used as earlier (Choubisa, 1988). <strong>The</strong><br />
control tissues were also run simultaneously to ensure<br />
the correct localization of these enzymes.<br />
<strong>The</strong> intensity of the activity of lipase, acid phosphatase,<br />
β-glucuronidase and simple esterase in different tissues<br />
of E. tewarii and C. udaipuriensis, as well as in rediae of<br />
E. tewarii has been depicted in Table I and II. Only<br />
intestinal ceaca of E. tewarii cercariae revealed the low<br />
intensity of acid phosphatase and simple esterase<br />
activity, whereas the caeca of C. udaipuriensis did not<br />
show the activity of the above mentioned enzymes.<br />
However, the gut of redia of E. tewarii was found to<br />
have maximum activity of acid phosphatase and<br />
esterase. <strong>The</strong> oesophageal gland of the present redia;<br />
however, was found to be positive <strong>for</strong> glucuronidase,<br />
lipase, esterase and acid phosphatase. <strong>The</strong> intense<br />
activity of lipase was observed on the body wall,<br />
cystogenous and penetration glands of echinostome<br />
cerariae, E. tewarii (Plate 1; Fig. 3), whereas the<br />
funcocercous cercariae also revealed the lipase activity<br />
but moderate in the penetration and cystogenous<br />
glands, body wall and excretory bladder (Plate 2; Fig. 4<br />
and 5). <strong>The</strong> oral suckers of both cercariae also indicated<br />
low activity of this enzyme. <strong>The</strong> activity of acid<br />
phosphatase was seen in both types of glands,<br />
penetration and cystogenous, as well as in excretory<br />
bladder of both cercarial species. Low activity of this<br />
enzyme was seen in intestinal ceaca only in the E.<br />
tewarii (Plate 1; Fig. 2). Whereas β−glucuronidase<br />
activity was not found in any part of the cercariae, on<br />
the contrary, the esterase was seen in the from of bluish<br />
micro-granules in various parts of cercarial body, its<br />
maxium activity was observed in the nervous system<br />
-4<br />
(Fig. 1 in both Plate 1 and 2). <strong>The</strong> use of 10 M eserine<br />
sulphate inhibited esterase activity completely in the<br />
nervous system and to a great extent in cystogenous and<br />
penetration glands, near ceaca. Except simple esterase<br />
activity, no other enzymes viz., acid phosphatase, lipase<br />
and β−glucuronidase revealed any activity in the tail of<br />
both types of cercariae.<br />
Table I. <strong>The</strong> activity of enzymes in redia and an unreleased cercariae, Echinostoma tewarii<br />
Enzymes Cercaria (E. tewarii) Redia of E. tewarii<br />
BW IC EB NS S T OG C<br />
Acid phosphatase + + ++ - + + +++ +++<br />
Lipase +++ - - - + - +++ -<br />
β-glucuronidase - - - - - - +++ -<br />
Simple esterase ++ + - +++ ++ ++ +++ +++<br />
+++, intense activity; ++, moderate activity; +, low activity; -, no activity;<br />
BW, body wall; C, caecum; EB, excretory bladder; IC, intestinal caeca; NS, nervous system; OG, oesophageal gland; S,<br />
suckers; T, tail.<br />
Table II. <strong>The</strong> activity of enzymes in an unreleased furcocercous cercariae, C. udaipuriensis<br />
Enzymes<br />
Cercaria (C. udaipuriensis)<br />
BW IC EB NS S T<br />
Acid phosphatase + + ++ - + -<br />
Lipase ++ - - - + -<br />
β-glucuronidase - - - - - -<br />
Simple esterase ++ - - +++ ++ +++<br />
+++, intense activity; ++, moderate activity; +, low activity; -, no activity;<br />
BW, body wall; C, caecum; EB, excretory bladder; IC, intestinal caeca; NS, nervous system; OG, oesophageal gland;<br />
S, suckers; T, tail.
70 Choubisa<br />
OS<br />
CC<br />
Oe<br />
PLNC<br />
ExB<br />
CR<br />
ANC<br />
PVNC<br />
VS<br />
OS<br />
CyG<br />
VS<br />
ExB<br />
Fig. 1. Localization of simple esterase in the cercaria, E. tewarii<br />
revealing intense activity in the nervous system and moderate in<br />
body wall, suckers and tail region (x 100).<br />
Fig. 3. E. tewarii cercaria revealing the intense activity of lipase<br />
in the body wall, cystogenous, pentration glands and in suckers.<br />
(x 100).<br />
Oe<br />
M PhG<br />
G<br />
C<br />
OS<br />
G<br />
M<br />
CyG<br />
Oe<br />
Ph<br />
VS<br />
ExB<br />
G<br />
C<br />
Fig. 2. Activity of acid phosphatase in the cercaria E. tewarii<br />
showing moderate activity in the excretory bladder,<br />
cystogenous and penetration glands and low in the body wall,<br />
suckers and intestinal caeca. (x 100).<br />
Fig. 4 and 5. Rediae of E. tewarii revealing the intense activity of<br />
simple esterase (Fig. 4) in the oesophageal gland and gut. (x<br />
100).
Mode of nutrition in pathogenic trematode larvae<br />
71<br />
OS<br />
CC<br />
PLNC<br />
PVNC<br />
CyG<br />
OS<br />
IC<br />
T<br />
ExB<br />
FR<br />
(Fig. 1) β-glucuranidase<br />
(Fig. 3) and lipase<br />
OS<br />
OS<br />
ExB<br />
FR<br />
IC<br />
Fig.4<br />
T<br />
OS<br />
IC<br />
ExB<br />
T<br />
(Fig. 2), acid phosphatase<br />
(Fig.4 and 5) activity in various regions.
72 Choubisa<br />
From the present study, it is evident that rediae of E.<br />
tewarii feed upon the hepatopancreatic tissue. <strong>The</strong> cell<br />
debris of the pancreas has been observed in the caecum<br />
of the rediae. Histology of the pathological tissue<br />
clearly supports tissue feeding of rediae.<br />
Certain hydrolytic enzymes such as acid phosphatase,<br />
ATPase and simple esterase have revealed their<br />
presence in the gut of the rediae in the present study. <strong>The</strong><br />
oesophageal gland of the redia; however, was found<br />
positive <strong>for</strong> lipase, -glucuronidase, esterase and acid<br />
phosphatase enzymes. Because no activity of lipase and<br />
-glucuronidase was observed in the caecum, it appears<br />
that these two enzymes are specially secreted by<br />
oesophageal glands <strong>for</strong> extracellular digestion of the<br />
food, the host tissues.<br />
Results of the present investigation corroborate the<br />
findings of Cheng (1964) and Probert (1966), who have<br />
also reported the ceacal wall to be very rich in acid<br />
phosphatase and non-specific esterase in their studies<br />
on rediae. <strong>The</strong> presence of phosphatases in the ceacal<br />
gut may not be directly involved in the digestion and<br />
absorption of nutrients, in view of the strong criticism<br />
by Reed (1966). Nevertheless, presence of cellular<br />
debris and multitude of digestive enzymes conclusively<br />
prove the redial gut to be an active site <strong>for</strong> the digestion<br />
of host cells.<br />
Although the redia in this study possesses a gut and a<br />
battery of enzymes <strong>for</strong> the digestion of a variety of<br />
nutrients such as proteins, glycogen and lipids present<br />
in the host tissues and taken as the food material, it does<br />
not preclude the possibility of obtaining the nutrients<br />
through tegument, as do the sporocysts and adult flukes.<br />
This contention gets support from the presence of<br />
phosphatases on the tegument of redia, as well as, the<br />
ultrastructural studies of the redia of Parochis acanthus<br />
(Rees, 1966) and Neophasis lageni<strong>for</strong>mis (Koie,<br />
1971a), in which their external tegument revealed the<br />
presence of microvilli and intense surface folding,<br />
respectively. Thus the body wall of the present redia<br />
may be regarded as analogous to the surface<br />
amplification that occurs at the outer surface of tape<br />
worms and sporocysts (Whitfield, 1979).<br />
When the echinostome and furcocercous cercariae of<br />
the present study were tested <strong>for</strong> the histochemical<br />
distribution of various hydrolytic enzymes in tegument<br />
and gut, it was found that except acid phosphatase no<br />
other enzyme gave positive activity. <strong>The</strong> absence of an<br />
array of enzymes in the gut, except acid phosphatase,<br />
clearly indicated the absence of digestion of food in this<br />
larval stage. <strong>The</strong> absence of most digestive enzymes in<br />
the caecae of cercariae are conducive to their being a<br />
non-feeding stage. Koie (1971b) has, however, reported<br />
the histochemical presence of some hydrolytic<br />
enzymes in the intestinal cells of the cercaria<br />
Zoogonoides viviparous, whereas Stirwalt and Walters<br />
(1964) have demonstrated aminopeptidase activity in<br />
the intestinal caeca of Schistosoma mansoni cercariae.<br />
<strong>The</strong> latter has, however, not discussed the significance<br />
of the enzyme in relation to larval nutrition.<br />
Acid phosphatase has been established as a marker <strong>for</strong><br />
lysosome (de Duve, 1963), and the lysosomes play an<br />
important role in the intracellular as well as<br />
intercellular digestion of several flat worms. Because<br />
cercariae in the present study are non-feeding larval<br />
stages and their caecal cells contain acid phosphatase,<br />
it is possible that the caecal cells during emergency<br />
(i.e., when glycogen reserve of the cercariae has<br />
exhausted) undergo sequestration in order to provide<br />
the threshold energy <strong>for</strong> the survival of the cercariae<br />
till it has reached the host and started obtaining<br />
nutrition from there. A similar suggestion has been<br />
made by Sharma and Mandawat (1982) <strong>for</strong> high<br />
concentration of acid phosphatase present in the<br />
caecal cells of the starving Ganeo tigrina recovered<br />
from hibernating frog host.<br />
Tegument of cercariae appears to compensate the<br />
function of the uptake of nutrients by non-functional<br />
intestinal caecae. In view of this contention, occurrence<br />
of certain hydrolytic enzymes such as acid phosphatase,<br />
lipase, -glucuronidase and simple esterase in the<br />
tegument of unreleased as well as free living cercariae<br />
E. tewarii and C. udaipuriensis are significant from<br />
permeability of nutrients point of view. <strong>The</strong>se results<br />
are similar to the findings of the other investigators who<br />
have advocated the role of phosphatases in the<br />
translocation of nutrients through the tegument of the<br />
cercariae of Zoogonoides viviparus, S. mansoni and<br />
Cloacitrema narrabeensis etc. (Stirewalt and Walters,<br />
1964; Dixon, 1970; Koie, 1971a). Although cercariae<br />
are advanced over rediae but from nutritional point of<br />
view this appears to be vice versa.<br />
<strong>The</strong> present study on the nutrition of redia and cercariae<br />
delineated a sharp contrast. <strong>The</strong> comparison revealed<br />
the redia to be nutritionally advanced over cercaria.<br />
And this seems to be an excellent adaptation and high<br />
strategy on the part of redia to obtain more nutrition so<br />
that more protein and other nutrients are made available<br />
to the developing cercariae within the body of the redia.
Mode of nutrition in pathogenic trematode larvae<br />
73<br />
ACKNOWLEDGEMENTS<br />
<strong>The</strong> author is grateful to Prof. P. N. Sharma (Eminent<br />
Parasitologist) <strong>for</strong> astute advice and comments.<br />
Koie M. 1971a. On the histochemistry and ultrastructures of the<br />
redia of Neophasis lageni<strong>for</strong>mis (Lebour, 1910) (Trematoda,<br />
Acanthocolpidae). Ophelia 9:113-143.<br />
Koie M. 1971b. On the histochemistry and ultrastructure of the<br />
REFERENCES<br />
tegument and associated structures of the cercaira of<br />
Zoogonoides viviparus in the first intermediate host. Ophelia<br />
Cheng TC. 1964. Studies on phosphatase systems in 9:165-206.<br />
hepatopancreatic cells of the molluscan host of<br />
Echinoparyphium sp. and in the redia and cercaria of this<br />
trematode. Parasitol 54:73-79.<br />
Cheng TC and Yee HWF. 1968. Histochemical demonstration of<br />
amino peptidase activity associated with the intra-molluscan<br />
stages of Philophthalmus gralli Mathis and Leger. Parasitol<br />
58:473-480.<br />
Choubisa SL. 1988. Histological and histochemical observations<br />
on the digestive gland of Melanoides tuberculatus<br />
(Gastropoda) infected with certain larval trematodes and<br />
focus on their mode of nutrition. Proc <strong>Indian</strong> Acad Sci (Anim<br />
Sci) 97:251-262.<br />
Choubisa SL. 1989. Distribution of non-specific esterase in<br />
certain larval digeneans with a note on morphology of<br />
nervous system. Ind J Exp Biol 27:32-57.<br />
Choubisa SL and Sharma PN. 1985. Cercaria tewarii n. sp.<br />
(Echinostomatid cercaria) from fresh water snail,<br />
Indoplanorbis exustus (Deshayes). Bio-Sci Res Bull 1:50-53.<br />
Clark TS. 1968. Histochemical studies of glandular contents of<br />
two sp. of cercairae. Am J Med Tech 34:446-452.<br />
de Duve C. 1963. <strong>The</strong> Lysosome concept. In: A.V.S. de Renk and<br />
M. P. Camerson (Eds). CIBA Foundation Symposium on<br />
lysosomes. London. J.A. Churchill.<br />
Dixon KE. 1970. Absorption by developing cercariae of<br />
Cloacitrema nerabeenesis (Philophthalmidae). J Parasitol<br />
56:416-417.<br />
Erasmus DA. 1972. <strong>The</strong> Biology of Trematodes. Printed in Great<br />
Britain at the University Press, Belfast.<br />
Fripp PJ. 1966. Histochemical localization of β-glucuromidase<br />
in Schistosomes. Exp Parasitol 19:254-263.<br />
Porter CW and Hall JE. 1970a. Histochemistry of Cotylocerous<br />
cercaria I. Glandular complex in Plagioporus lepomis. Exp<br />
Parasitol 27:368-377.<br />
Porter CW and Hall JE. 1970b. Histochemistry of a Cotylocerous<br />
cercaria II. Hydrolytic and oxidative enzymes in<br />
Plagioporous lepomis. Exp Parasitol 27:378-387.<br />
Probert AJ. 1966. Histochemical studies on the rediae and<br />
cercariae of Echinoparyphium recurvatum Linstow. Nature<br />
210:550-551.<br />
Reed CP. 1966. Nutrition of intestinal helminths. In : Biology of<br />
Parasites. EJL Soulsby (Ed.), Academic Press. pp. 101-126.<br />
Rees FG. 1966. Light and electron microscope studies of the<br />
redia of Parorchis acanthus. Nicoll 56:589-602.<br />
Sharma PN and Mandawat S. 1982. A comparison of<br />
morphology in Ganeo tigrinum from hibernating and nonhibernating<br />
Rana cyanophyctis and R. tigrina. J Helminthol<br />
56:5-10.<br />
Sharma PN and Choubisa SL. 1983. Cercaria udaipuriensis n.sp.<br />
from fresh water snails, Vivipara bengalensis from Fateh<br />
Sagar lake. Ind J Parasit 7:209-212.<br />
Stirewalt MA and Walters M. 1964. Histochemical assay of<br />
glands of cercarial of Schistosoma mansoni. J Parasitol 50<br />
Suppl. No. 94:44.<br />
Whitfield PJ. 1979. <strong>The</strong> Biology of Parasitism: An introduction<br />
to the study of associating organisms. Edward Arnold<br />
(Publisher).
Journal of Parasitic Diseases: <strong>June</strong> <strong>2008</strong>, Vol. 32, No. 1, 74-76<br />
Short communication<br />
J P D<br />
Amoebic dysentery in dogs and dog owners<br />
1 2 2 3<br />
Saleh Umair , Azhar Maqbool , Zubair Shabbir and M. D. Ahmad<br />
1<br />
Department of <strong>Parasitology</strong>, University of Veterinary and Animal Sciences, Lahore, Pakistan.<br />
2<br />
University Diagnostic laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan.<br />
3<br />
Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore, Pakistan.<br />
Received 15 September <strong>2008</strong>; revised 19 September <strong>2008</strong>; accepted 27 September <strong>2008</strong><br />
ABSTRACT. A total of 100 samples each of faeces and serum of dogs, and of their owners were<br />
collected <strong>for</strong> the detection of Entamoeba histolytica. E. histolytica, in feces, was examined directly as<br />
well as by flotation method, whereas in case of extra luminal invasion their antibody level was<br />
determined by latex agglutination test. <strong>The</strong> prevalence of E. histolytica in faeces of dogs and humans<br />
was estimated to be 14% and 10%, respectively. <strong>The</strong> prevalence of extra luminal amoebiasis, as<br />
determined by latex agglutination test which detected the antibodies in serum of dogs and their<br />
owners, was estimated to be 12% and 8%, respectively.<br />
Keywords: amoebiasis, latex agglutination, prevalence<br />
<strong>The</strong> best known zoonotic species of amoebae<br />
parasitizing humans is Entamoeba histolytica, the<br />
causative agent of amoebic dysentery or amoebiasis.<br />
An estimated 400–500 million people in the world are<br />
infected with E. histolytica, and only 5% to 10% of<br />
them show symptoms of the disease (Garcia and<br />
Bruchner 1997). <strong>The</strong> usual mode of E. histolytica<br />
infection is the ingestion of cysts from contaminated<br />
hands, food or water, and the incidence of the disease<br />
increase considerably in thickly populated areas where<br />
contact with infected individuals is more likely. E.<br />
histolytica exhibits a typical fecal-oral life cycle<br />
consisting of infectious cysts passing out in the faeces,<br />
and trophozoites which replicate within the large<br />
intestine. <strong>The</strong> infection is acquired through the<br />
ingestion of cysts, and the risk factors are similar to<br />
those of other diseases transmitted by the fecal-oral<br />
route; contaminated food and water are probably the<br />
primary sources of infection. <strong>The</strong> higher prevalence in<br />
Corresponding author: Prof. Dr. Azhar Maqbool, Department<br />
of Parasitilogy, University of Veterinary and Animal Sciences,<br />
Lahore, 54000, Pakistan. E-mail: salehumair@hotmail.com<br />
areas of lower socioeconomic status is likely due to<br />
poor sanitation and a lack of indoor plumbing.<br />
However, a higher prevalence of E. histolytica infection<br />
is also observed in institutions such as mental hospitals,<br />
orphanages and prisons, where crowding and problems<br />
with fecal contamination are the contributing factors.<br />
<strong>The</strong> symptoms of amoebiasis range from mild<br />
abdominal discom<strong>for</strong>t with bloody mucous diarrhoea<br />
alternating with periods of constipation or remission to<br />
acute or fatal dysentery with fever, chill and bloody or<br />
mucoid diarrhoea (Benenson, 1995). Dissemination<br />
can carry the parasite to the other vital organs like liver,<br />
where they produce focal necrosis.<br />
In view of the zoonotic importance of amoebiasis, the<br />
present study was designed to study the prevalence of<br />
amoebiasis in dogs and their owners by using rapid<br />
latex agglutination test, flotation method and direct<br />
faecal examination.<br />
Determination of the prevalence of E. histolytica<br />
Collection of faecal samples: A total of 100 faecal<br />
samples (dogs, n = 50; humans, n = 50) were examined<br />
<strong>for</strong> amoebiasis. <strong>The</strong>se samples were collected from Pet
Amoebic dysentery in dogs<br />
75<br />
centre, University of Veterinary and Animal Sciences, vary according to socio cultural habits, geographic<br />
Lahore and Lahore Animal Hospital. History of dogs factors, climate and route of transmission. In the present<br />
and their owners was regularly noted in prescribed study, an overall 11% prevalence of anti-Entamoeba<br />
pro<strong>for</strong>ma. For faecal diagnosis of E. histolytica, both antibodies was detected. In dogs, a 14% faecal<br />
direct faecal examination and flotation method were prevalence was detected, whereas in man it was 10 %.<br />
used, as described by Soulsby (1982). Serum prevalence of dog and man was 12% and 10 %,<br />
respectively. Many workers from different parts of the<br />
Collection of Serum Samples: A total of 100 serum<br />
world have reported the prevalence of anti-Entamoeba<br />
samples (dogs, n = 50; humans, n = 50) were tested <strong>for</strong><br />
antibodies by using various serological techniques<br />
amoebiasis. Under aseptic measures, 8–10 ml volumes<br />
including latex agglutination test (LAT), e. g. Rizwan<br />
of blood were drawn by veinipuncture with the help of<br />
et al. (1999) have reported that 15-20% dog owners<br />
disposable syringes and transferred slowly to screwfrom<br />
Karachi (Pakistan) and 12 % pet owners in<br />
capped sterile test tubes, to avoid hemolysis (Benjamin,<br />
Tanzania (Gilli et al., 1992) had anti-Entamoeba<br />
1985). Serum samples were then separated by first<br />
antibodies in their serum. However, highest (35 %)<br />
clotting the blood and then centrifugation at 3500 rpm<br />
prevalence has been reported by Abioye (1972) in<br />
<strong>for</strong> 5 min. All the collected serum samples were<br />
Ibadan. This variation in the results could be due to<br />
analyzed <strong>for</strong> specific Entamoeba antibodies by using<br />
difference in environmental and geographical<br />
latex agglutination test. For this purpose, the<br />
conditions. It is interesting to note that pet keeping led<br />
commercial test kit “Entamoeba Latex”, manufactured<br />
to increase in the incidence of amoebiasis (Anand et al.,<br />
by Quimica Clinica Apelicada, SA Asmota, Spain, was<br />
1997). Further, it has been observed that if a dog owner<br />
used.<br />
is infected with amoebiasis, his dog is also found to be<br />
Amoebiasis is a disease that is dispersed worldwide, infected. It may thus mean that, most probably, the dog<br />
and its seropositivity levels widely vary in different owners could contract infection from their dog.<br />
regions of the globe (David 1972). <strong>The</strong> prevalence rates Curiously, amoebiasis is age related as its prevalence is<br />
Table I. Distribution of E. histolytica in humans determined by using latex aagglutination, direct fecal examination<br />
method and flotation method<br />
Sample No. Anti body Direct Flotation % % Fecal<br />
tested titer fecal Seropositive<br />
reciprocal<br />
method<br />
16 128 256<br />
Serum of dog 50 1 1 2 - - 8 -<br />
owners<br />
Fecal of 50 4 5 10<br />
dog owners<br />
Total 100 1 1 2 4 5 8 10<br />
Table II. Distribution of E. histolytica in various age groups of dogs by using latex agglutination test<br />
Age group No. tested Reciprocation Seropositive %<br />
(years)<br />
Seropositive<br />
1:16 1:128 1:256<br />
0-2 06 1 1 16.6<br />
3-4 15 1 1 1 3 20<br />
5-6 16 1 1 6/23<br />
7 and above 13 1 1 7.2<br />
Total 50 2 2 2 6 12
76 Umair et al.<br />
Table III. Distribution of E. histolytica in various age groups of dogs by using direct fecal and flotation method<br />
Age groups No. Positive % Positive Positive % Positive<br />
(years) tested fotation flotation direct direct<br />
method method method method<br />
0-2 06 1 16.6 0 0<br />
3-4 15 4 26.1 3 20<br />
5-6 16 1 6.23 1 6.23<br />
7 and above 13 2 15.4 2 15.4<br />
Total 50 8 16 6 12<br />
higher in animals of younger age. Dogs between 1–3<br />
years of age are known to be maximally susceptibility<br />
to amoebiasis.<br />
REFERENCES<br />
Abioye AA and Edington GM. 1972. Prevalence of amoebiasis at<br />
autopsy in Ibadan. Transactions of the Royal <strong>Society</strong> of<br />
Tropical Medicine and Hygiene 5:754-763.<br />
Anand AC, Reddy PS, Saiprasad GS, Kher SK. 1997. Does nondysenteric<br />
intestinal amoebiasis exist? <strong>The</strong> Lancet 9045:89-<br />
92.<br />
Benjamin MM. 1985. Outline of Veterinary Pathology III (Ed.)<br />
Kalyani Publishers, New Delhi. pp. 102-104.<br />
David Botero R. 1972. Studies on the stability of the<br />
hemagglutinating antibody of Entamoeba histolytica.<br />
Transactions of the Royal <strong>Society</strong> of Tropical Medicine and<br />
Hygiene 66: 517-518.<br />
Gilli S, Petithory JC, Adoin F, Pannetier C and Scaglia M. 2001.<br />
Asymptomatic amoebic infection: Entamoeba histolytica or<br />
Entamoeba dispar. Bulliten of <strong>Society</strong> of Pathology Exotics<br />
94:304-307.<br />
Gómez Maganda T, García Carrizosa R, Torres Valadez F, Ortiz<br />
Ramírez E, Villaseñor de la Parra C , Flores González A and<br />
Gómez García E. 1978. Latex agglutination test in amebic<br />
liver abscess. Revista de Gastroenterologia de Mexico 43:21-<br />
28.<br />
Morris MN, Powell SJ and Elsdon-Dew R. 1971. Invasive<br />
amoebiasis: circulating antibody levels by latex agglutination<br />
test. South African Medicine Journal 45:1206-1208.<br />
Rizwan, QM, Syed A and Hafiz A.1999. Acute and chronic<br />
amoebiasis. <strong>The</strong> professional 6:364-371.<br />
Soulsby EJL. 1982. Helminths, Arthropods and Protozoa of<br />
Domestic Animals. (Ed.). Bailliere Tindall, London, UK. Pp.<br />
765-766.
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significance. In no case it should be just a statement of<br />
results only. References are not allowed in the<br />
abstract.<br />
Keywords: 3-5 keywords arranged in alphabetical<br />
order should be provided below the abstract.<br />
Introduction: Introduction should provide the<br />
essential background, must briefly review the relevant<br />
literature, identify the question(s) to be answered and<br />
should state the reasons <strong>for</strong> undertaking the study.<br />
Further, to avoid suspense, it should end with a brief<br />
statement of results.<br />
Material and Methods: <strong>The</strong> source of the material<br />
used should be clearly mentioned. <strong>The</strong> procedures<br />
adopted should be explicitly stated to enable other<br />
workers to reproduce the results. New methods may be<br />
described in sufficient details, established methods<br />
can be just described in brief with appropriate<br />
reference(s). Deviations from previously reported<br />
methods, if any, should be explained giving the<br />
reasons thereof.<br />
ii
Results: <strong>The</strong> results should be as clear as possible and •Cheever AW, Macedonia JG, Mosimann JE and<br />
must state only the findings of the study reported. <strong>The</strong>y Cheever EA. 1994. Kinetics of egg production<br />
should be comprehendible to the readers and and egg excreted by Schistosoma mansoni and S.<br />
sequenced in such a way that a logical progression is japonicum in mice infected with a single pair of<br />
developed. <strong>The</strong> data presented in figures and tables worms. American Journal of Tropical Medicine<br />
should not be repeatedly summarized. Results should and Hygiene. 50:281-285.<br />
not be intermixed with interpretations or conclusions.<br />
<strong>The</strong> name of the journal should either be given in full<br />
Discussion: In this section the results should be<br />
discussed in a focused way and not just recapitulated.<br />
or abbreviated as per the Index Medicus or Medline.<br />
Authors should try to present the principles, Edited books<br />
generalizations and relationships. <strong>The</strong>y should<br />
specifically show how the results and interpretations<br />
Schaniz PM and Kagan IG. 1980. Echinococcosis. In:<br />
reported in the manuscript agree or disagree with<br />
Immunological Investigations of Tropical Parasitic<br />
already published in<strong>for</strong>mation. <strong>The</strong> significance and<br />
Diseases. Houba. V (Ed.), Churchill Living stone,<br />
importance of the work must also be discussed<br />
New York. pp 104-129.<br />
sufficiently. Conclusions must be stated clearly and<br />
evidence <strong>for</strong> each conclusion should be summarized.<br />
Conference proceedings<br />
Long, rambling discussion lacking logic and flow Singh, Prati Pal and Mortensen RF. 1985. Serum<br />
should be avoided. Speculations must be avoided to amyloid P-component (SAP) response in inbred<br />
the extent possible.<br />
mouse strains during Listeria monocytogenes<br />
infection. In: Proceedings of II International<br />
Acknowledgements: Acknowledgements should be<br />
Symposium on Genetic Control of Host Resistance to<br />
brief and made <strong>for</strong> specific scientific, technological<br />
Infection and Malignancy, Montreal (Ed. E.<br />
and monetary (grant) assistance.<br />
Skamene), Allan R. Liss, Inc., New York, pp 339-344.<br />
References: List of references should include only<br />
published papers and papers in press. Abstracts<br />
Reports<br />
presented in meetings will not be considered as World Health Organization. 1984. <strong>The</strong> Leishmaniasis.<br />
references. Citations of unpublished work, where Technical Report Series No. 701. Geneva,<br />
absolutely necessary, should be incorporated in the Switzerland.<br />
text itself (examples: our unpublished observations or<br />
Sharma et al. unpublished data or Sharma et al. Electronic <strong>for</strong>mat papers<br />
personal communications). Personal communications<br />
must be supported by an authorized letter of Ponce LV. 2005. Adoptive transfer of dendritic cells<br />
permission. References in the text should be cited modulates immunogenesis and tolerogenesis in a<br />
according to the name and year system (also known as neonatal model of murine cutaneous leishmaniasis.<br />
Harvard system) in chronological order, using 'and' <strong>for</strong> Kinetoplastid Biology and Disease. 4:2.<br />
articles with two authors and et al. <strong>for</strong> those with more http://www.kinetoplastids.com/content/4/1/2 .<br />
than two authors and semi colon between references<br />
Papers published only with Digital Object Identifier<br />
(Trager and Jensen, 1976; Sharma et al., 1994).<br />
(DOI) numbers<br />
References should be listed at the end in alphabetical<br />
order and it must be ensured that all references cited in Jabakhanji R. Inflammatory and neuropathic pain<br />
the text are included in the list and vice versa. <strong>The</strong> animals exhibit distinct responses to innocuous and<br />
following style should be followed:<br />
motoric challenges. Molecular Pain. DOI:10.1186/<br />
Journal<br />
1744-8069-2-1.<br />
In the list of references at the end of the paper, all the<br />
names of authors should be given. However, if there<br />
are more than six authors, list first six names followed<br />
by et al.<br />
•Trager W and Jensen JB. 1976. Human malaria<br />
parasite in continuous culture. Science.<br />
193:673-675.<br />
iii<br />
Tables: Tables should be typed separately and<br />
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headings. Statistical variations such as SD and SE<br />
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journal does not follow floating number principle, so Interleukins: Designations recommended by IUIS<br />
write 1 and not 1.0.<br />
committee on interleukins have to be used (European<br />
Cytokine Network. 1991. vol. 2 (5). pp 309-310).<br />
Illustrations: Three sets (one original and two firstclass<br />
copies) of illustrations should be submitted, Abbreviations: Only standard abbreviations are to be<br />
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iv
JOURNAL OF PARASITIC DISEASES<br />
(Official organ of the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong>)<br />
Undertaking by Authors<br />
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We, the undersigned, give an undertaking to the following effect with regard to our manuscript titled:<br />
_____________________________________________________________________________________________<br />
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submitted <strong>for</strong> consideration to possible publication in the Journal of Parasitic Diseases.<br />
1. Research reported was conducted within the proper animal care and according to the applicable National Laws.<br />
2. <strong>The</strong> manuscript mentioned above has not been submitted <strong>for</strong> publication in any <strong>for</strong>m to any other journal.<br />
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Important<br />
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v
<strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
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India : Life Membership : Rs. 2000.00 Other Countries : US$ 400.00<br />
(Demand draft should be drawn in favour of Secretary, <strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong>, payable at Lucknow)<br />
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vi
THE INDIAN SOCIETY FOR PARASITOLOGY<br />
Executive Committee<br />
President<br />
Prof. Veena Tandon<br />
Department of Zoology<br />
North Hill University<br />
Shillong<br />
Vice-President<br />
Dr. J. Mahanta<br />
Regional Medical Research Centre<br />
Dibrugarh<br />
Vice-President<br />
Prof. P. D. Juyal<br />
Department of Veterinary <strong>Parasitology</strong><br />
Guru Angad Dev Vet. & Ani. Sci. Univ.<br />
Ludhiana<br />
Joint Secretary<br />
Dr. Vas Dev<br />
Malaria Research Center<br />
Sonapur<br />
Vice-President<br />
Prof. R. Madhvi<br />
Department of Zoology<br />
Andhra University<br />
Vishakapatnam<br />
Secretary<br />
Dr. J. K. Saxena<br />
Division of Biochemistry<br />
Central Drug Research Institute<br />
Lucknow<br />
Treasurer<br />
Dr. L. M. Tripathi<br />
Division of <strong>Parasitology</strong><br />
Lucknow<br />
Members<br />
Dr. Wasim Ahmed, Aligarh<br />
Dr. P. Prakash Babu, Hyderabad<br />
Dr. S. C. Datta, Kolkata<br />
Dr. S. K. Ghosh, Bangalore<br />
Dr. Neena Goyal, Lucknow<br />
Prof. Neelima Gupta, Bareilley<br />
Dr. B. V. Jadhav, Aurangabad<br />
Dr. A. M. Khan, Dibrugarh<br />
Dr. Ashwani Kumar, Goa<br />
Prof. G. G. Mani, Visakhapatnam<br />
Dr. S. K. Puri, Lucknow<br />
Prof. R. Kaleysa Raj, Trivandrum<br />
Dr. K. K. Saxena, Bareilley<br />
Prof. Prati Pal Singh, S. A. S. Nagar<br />
Statements and opinions expressed in the Journal of Parasitic Diseases or in the presentations<br />
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