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Volume 28<br />
No. 1<br />
June 2004<br />
ISSN : 0971-7196<br />
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Published By<br />
<strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
Electronic version available on ISP Website<br />
www.parasitologyindia.org
JOURNAL OF PARASITIC DISEASES<br />
(ISSN : 0971-7196)<br />
Patron<br />
Prof. M. Shamim Jairajpuri,<br />
President, <strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
&<br />
Professor, Department of Zoology Aligarh Muslim University, Aligarh - 202002, India<br />
Editor-in-Chief<br />
Prof. Nancy Malla,<br />
Department of <strong>Parasitology</strong><br />
PGIMER, Chandigarh<br />
Managing Editor<br />
Dr. M.L. Dubey<br />
Department of <strong>Parasitology</strong><br />
PGIMER, Chandigarh<br />
Dr. A.B. Chowdhury<br />
Kolkata<br />
Dr. N.K. Ganguly<br />
New Delhi<br />
Dr. V.S. Chauhan, New Delhi<br />
Dr. N.J. Shetty, Bangalore<br />
Dr. B.C. Harinath, Sevagram<br />
Dr. Durdana S. Jairajpuri, Aligarh<br />
Dr. Q.H. Baqri, Jodhpur<br />
Dr. D.P. Haldar, Kalyani<br />
Dr. Md. Hafeez, Tirupati<br />
Dr. V.M.L. Srivastava, Lucknow<br />
Dr. S.C. Parija, Pondicherry<br />
Advisory Board<br />
Dr. R.C. Mahajan<br />
Chandigarh<br />
Editorial Board<br />
Dr. V.P. Sharma<br />
New Delhi<br />
Dr. G.P. Dutta<br />
Lucknow<br />
Dr. Chetan Chitinis, New Delhi<br />
Dr. R. Madhavi, Vishakhapatnam<br />
Dr. A.R. Khan, Srinagar<br />
Dr. Sobhna Sharma, Mumbai<br />
Dr. Buddhadev Mana, Kolkata<br />
Dr. Ch. Dhanachand, Manipur<br />
Dr. Jagdish Mahanta, Dibrugarh<br />
Dr. B. Jadhav, Aurangabad<br />
Dr. R. Madhubala, New Delhi<br />
Journal of Parasitic Diseases is published biannually by the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong> in June and December<br />
in each calender year. <strong>The</strong> subscription price <strong>for</strong> libraries and other multi reader organizations <strong>for</strong> each number is Rs. 400 in<br />
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to Dr. J.K. Saxena, Secretary, <strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong>, Division of Biochemistry, Central Drug Research Institute,<br />
Chattar Manzil, Lucknow - 226 001 (India).<br />
<strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
Executive Committee<br />
(2002-2004)<br />
President<br />
Prof. M.S. Jairajpuri, Aligarh<br />
Vice-Presidents<br />
Dr. J. Mahanta, Dibrugarh<br />
Dr. V.M.L. Srivastava, Lucknow<br />
Secretary<br />
Dr. J.K. Saxena, Lucknow<br />
Jt. Secretary<br />
Dr. T. Adak, New Delhi<br />
Treasurer<br />
Dr. L.M. Tripathi, Lucknow<br />
Members<br />
Prof. Irfan Ahmad, Aligarh<br />
Prof. Q.H. Baqri, Jodhpur<br />
Dr. A.P. Dash, Bhubaneshwer<br />
Dr. Neeru Singh, Jabalpur<br />
Prof. N.J. Shethy, Bangalore<br />
Dr. V.S. Chauhan, New Delhi<br />
Dr. Prakash Babu, Hyderabad<br />
Prof. Kaleysa Raj, Trivandrum<br />
Dr. B.V. Jadhav, Aurangabad<br />
Dr. Neelima Gupta, Bareilly<br />
Statements and opinions expressed in the Journal of Parasitic Diseases or in the presentations during the regular<br />
meeting of the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong> are those of the author(s) and do not necessarily reflect the official position of<br />
the <strong>Society</strong>. <strong>The</strong> Editorial Board, Publisher and the <strong>Society</strong> disclaim any responsibility <strong>for</strong> the accuracy of statements made<br />
by the contributors.<br />
Copyright ©2004 <strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong>
JOURNAL OF PARASITIC DISEASES<br />
Volume 28, Number 1, June 2004<br />
OFFICIAL ORGAN OF<br />
THE INDIAN SOCIETY FOR PARASITOLOGY<br />
Head Office : Central Drug Research Institute, Lucknow - 226 001, India<br />
Editorial Office : Department of <strong>Parasitology</strong>, Post Graduate Institute of Medical Education and Research,<br />
Chandigarh-160 012 India<br />
Fax : 0172-2744401, E-mail : medinst@pgi.chd.nic.in
Journal of Parasitic Diseases<br />
Copyright © 2004 <strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
All Rights Reserved.<br />
No part of this publication may be reproduced or utilized in any <strong>for</strong>m or by any means, electronic or<br />
mechanical including photocopying, recording or any other in<strong>for</strong>mation storage and retrieval system<br />
without the permission in writing from copyright owner.
JOURNAL OF PARASITIC DISEASES<br />
Volume 28 Number 1 June 2004<br />
CONTENTS<br />
PRESIDENTIAL ADDRESS<br />
<strong>The</strong> Perfect Match - Parasite & host: Made <strong>for</strong> each other 1<br />
M.S. Jairajpuri<br />
REVIEW<br />
Role of DNA microarray Technology <strong>for</strong> understanding differential 5<br />
gene expression in Parasitic Diseases<br />
A.Debnath, A. Sen, James H. Mckerrow and P. Das<br />
EPIDEMIOLOGY<br />
Epidemiological studies on bovine microfilariasis in coastal districts 17<br />
of Andhra Pradesh<br />
V. Pavan Kumar, B. Sreedevi, T. Venkata Reddy<br />
and K. Nalini Kumari<br />
Seasonal occurrence of helminth parasites in Schizothorax in 23<br />
Dal Lake Kashmir<br />
A.R. Khan, M.Z. Chishti, Fayaz Ahmad, Majidah Rashid and<br />
Shafqat Bakshi<br />
DIAGNOSIS<br />
A monoclonal antibody to 120 kDa B. malayi antigen with 29<br />
diagnostic potential in Bancroftian filariasis<br />
Balaji Ganesh, B. Parab P.B., Katdare M., Reddy M.V.R.<br />
and Harinath B.C.<br />
CHEMOTHERAPY<br />
Anthelmintic efficacy of extract of Stephania glabra and aerial root 37<br />
extract of Trichosanthes multiloba in vitro: two indigenous plants in<br />
Shillong, India.<br />
V. Tandon, L.M. Lyndem, P.K. Kar, P.Pal, B. Das and H.S.P. Rao<br />
Anthelmintic efficacy of genistein, the active principle of 45<br />
Flemingia vestita (Fabaceae): Alterations in the activity of<br />
the enzymes associated with the tegumental and gastrodemal<br />
interfaces of the trematode, Fasciolopsis buski<br />
Pradip Kumar Kar and Veena Tandon
SHORT COMMUNICATION<br />
Prevalence of phthirapteran ectoparasitic insects on domestic hens 57<br />
of Rampur(U.P.)<br />
A.K. Saxena, Sandeep Kumar, Nidhi Gupta and S.K. Singh<br />
Stilesia daulatabadensis N. Sp. from Capra hircus 61<br />
VP Shelke and GB Shinde<br />
Editorial Policy & Instructions to Authors<br />
Undertaking by Authors<br />
Membership Application<br />
Forthcoming Scientific Events
Journal of Parasitic Diseases<br />
Vol. 28 (1) June, 2004, pp. 1-4<br />
Presidential Address<br />
3rd Global Meet on Parasitic Diseases<br />
(January 12-16, 2004, Bangalore)<br />
<strong>The</strong> Perfect Match<br />
Parasite & Host : Made <strong>for</strong> each other<br />
M. Shamim Jairajpuri<br />
President, <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
Department of Zoology, Aligarh Muslim University<br />
Aligarh -202002, India<br />
he Darwin's <strong>The</strong>ory of Evolution states that<br />
Tevery species produces excessively large number<br />
of offsprings much more than their own existing<br />
populations. But the Natural Selection allows only<br />
those individuals with suitable variations to survive.<br />
<strong>The</strong> ecosystem, however, requires population<br />
constancy of the entire component species. This raises<br />
the important question as to how does the Nature<br />
manage these two rather contrasting phenomena?<br />
Further, what is the actual mechanism by means of<br />
which the unwanted rise in the population level is<br />
managed and brought down? <strong>The</strong> answer is that it is the<br />
parasites, pathogens, parasitoids, pests and the<br />
predators which may be called as 5 'Ps' that carry this<br />
out and <strong>for</strong> this reason they are often regarded as<br />
"Nature's Hangmen". <strong>The</strong>se organisms thus play a<br />
very significant role in population balance and they<br />
have been per<strong>for</strong>ming this duty ever since this living<br />
world has come into existence.<br />
<strong>The</strong> food is basic necessity <strong>for</strong> all organisms. Various<br />
feeding types and interactions have evolved between<br />
different animal and plant species during the course of<br />
evolution. <strong>The</strong>se may be microphagous,<br />
saprophagous, phytophagous, carnivorous,<br />
predaceous, etc. In parasitism, species or group of<br />
organisms called parasites do not have the ability to<br />
obtain food directly from the ecosystem; consequently<br />
they have to depend on some other organisms (host)<br />
<strong>for</strong> food. <strong>The</strong> physiological compatibility between the<br />
two is such that quite often the host and the parasite coevolve<br />
though not necessarily at the same pace. <strong>The</strong><br />
parasite-host interactions may occur among animal-<br />
animal-plant-plant and these provide some very<br />
interesting associations in different types of<br />
organisms. Consequently, there is a need to look into a<br />
few of these aspects.<br />
All organisms prefer to live in peace and harmony in<br />
their ecosystems. However, <strong>for</strong> their survival and to<br />
obtain food they may be responsible <strong>for</strong> causing death<br />
of others, if the situation so demands. But this is a part<br />
of Nature's overall strategy <strong>for</strong> maintaining an<br />
ecological balance. <strong>The</strong> host-parasite associations are<br />
unique in the sense that of the two organisms it is only<br />
the parasite that is benefitted while the host suffers.<br />
However, the parasite also does not gain in anyway by<br />
the death of its host because this amounts to destroying<br />
its own source of food. <strong>The</strong> perfect situation, to the<br />
advantage of the parasite, would indeed be to obtain its<br />
food without causing any serious or significant<br />
damage to the host. Such an ideal situation is not<br />
always known to exist but theoretically all parasitehost<br />
associations ultimately progress in this direction.<br />
It is precisely due to this reason that perfectly sound<br />
host-parasite combinations of species co-evolve and<br />
co-exist. Further, closely related species of hosts are<br />
known to possess very similar to often identical<br />
species of parasites. In developing harmony with the<br />
host, the parasite species also has to synchronize and<br />
adapt itself to a great extent not only structurally and<br />
ecologically but physiologically and biochemically as<br />
well. In making adjustments to suit each other, the host<br />
and the parasite become a perfect match leading to<br />
synchronization in their adult and larval stages of<br />
development. But the term matching or made <strong>for</strong> each
2 Jairajpuri : Presidential Address<br />
JPD : Vol. 28 (1), 2004<br />
other should not lead someone to believe that the<br />
parasitism in anyway is synonymous either with<br />
commensalism or symbiosis. In the latter association<br />
actually none of the organisms involved suffer any<br />
kind of disadvantage or injury. <strong>The</strong> same is not true in<br />
parasitism as the host species definitely suffers<br />
varying degrees of injury, which may be insignificant<br />
or temporary inconvenience to some sort of discom<strong>for</strong>t<br />
or disease which may even lead to the death of the host<br />
organism.<br />
It is evident that the adult stages of the parasite no<br />
doubt adapt themselves to a life in or on their host and<br />
in due course of time gradually attain a state of<br />
equilibrium with them. Of special importance are the<br />
life stages (young ones/larvae/juveniles) of the<br />
parasites and the various strategies that these may<br />
adapt to gain access/entry into their hosts. Some of this<br />
could be done directly but in others an intermediate<br />
host, a vector or carrier may be involved. An extreme<br />
kind of parasitism is exhibited by the parasitic<br />
crustacean, Sacculina which initially has a nauplius<br />
larva much like any other species of Crustacea but this<br />
soon changes into a cypris larva with a bivalve shell<br />
like that of the cirripedes. This larva attaches itself to<br />
one of the setae of the crab host and it soon throws off<br />
its limbs and becomes reduced to a mass of<br />
undifferentiated cells enclosed in the skin. This results<br />
into total loss of all organs of the parasite except the<br />
reproductive. For feeding, Sacculina develops rootlike<br />
processes which penetrate into various organs of<br />
the crab body. It is evident that the parasites in order to<br />
accommodate themselves to their host make great<br />
adjustments. In the present case, Sacculina has<br />
sacrificed its total identity.<br />
An interesting and quite noteworthy correlation exists<br />
in the life history of the monogenetic trematode,<br />
Polystoma integrrimum with its host, the frog. <strong>The</strong><br />
parasite attaches itself to the transparent walls of the<br />
urinary bladder of frogs. <strong>The</strong> fluke lays its eggs during<br />
the spring and its onchomiracidia are ready almost at<br />
the same time when the tadpoles of the frog are<br />
attaining the internal gill stage of their development.<br />
<strong>The</strong> little ciliated larva then gets attached to the<br />
tadpole. As the gills change into lungs and the frog<br />
moves towards adulthood, the fluke also becomes a<br />
young adult almost simultaneously and settles down in<br />
the bladder. This shows how beautifully the life cycles<br />
of the parasite and its host are matched. In case the<br />
onchomiracidia hatch early and get attached to the<br />
external gills of the tadpole, these become adults<br />
quickly, but outside the body of frog. Consequently,<br />
they perish without reaching the urinary bladder. In<br />
much the same way, the Protozoa Balantidium,<br />
Opalina and Nyctotherus which live inside the rectum<br />
of frog produce cysts only during spring time when the<br />
tadpoles are also present in the pond to feed upon them.<br />
This way the infection of the parasites passes on<br />
successfully from one generation of the host to the<br />
other.<br />
<strong>The</strong> filarial worms that parasitize human beings and<br />
animals provide one of the finest examples of<br />
synchronization in the life cycle between the host and<br />
its parasite. Wucheraria bancrofti, the Bancroft's<br />
filarial worm produces its young ones called<br />
microfilaria which appear in the peripheral blood<br />
circulation during the night when the host is asleep.<br />
This coincides with the timing of the visit of<br />
mosquitoes, the intermediate host. As mosquitoes bite<br />
and suck blood, the microfilaria pass into their bodies<br />
and subsequently develop into the infective stages.<br />
<strong>The</strong> latter are transferred back to man when the<br />
mosquitoes bite again <strong>for</strong> their blood meal. However,<br />
nocturnal periodicity would change into diurnal in<br />
those persons who work by the night and rest/sleep<br />
during the daytime. <strong>The</strong> same will happen, if an<br />
infected person happens to travel halfway round the<br />
globe. Another filarial worm called eye worm, Loa loa<br />
has diurnal periodicity because its intermediate host<br />
are day-biting tabanid flies, Chrysops sp. <strong>The</strong> fiery<br />
serpent, called the guinea or the Medina worm,<br />
Dracunculus medinensis is also a good example of a<br />
nematode species which parasitizes man. <strong>The</strong> females<br />
upon maturity contain millions of juveniles which are<br />
to be liberated in water so as to reach their intermediate<br />
host, the Cyclops. <strong>The</strong> females begin crawling towards<br />
the hand and feet, which are most likely to come in<br />
contact with water. Biologically, it is a very interesting<br />
phenomenon and one wonders as to how do these<br />
females know that their best chance of liberating the<br />
juveniles in water is to move towards extremities of the<br />
host. However, in those people who carry water on
JPD : Vol. 28 (1), 2004<br />
Jairajpuri : Presidential Address<br />
3<br />
its body. During evolutionary process and in order to<br />
mitigate the severity of the host-parasite struggle, both<br />
the parasite and the host make all possible ef<strong>for</strong>ts to<br />
adapt to each other. <strong>The</strong> parasite causing less and less<br />
damage and the host gradually getting used to the<br />
presence of the parasite. Consequently, equilibrium is<br />
reached in this association when the parasite inflicts<br />
minimum or no damage to the host.<br />
From the <strong>for</strong>egoing account let no one get the<br />
impression that I am eulogizing the parasites in<br />
anyway. What I have desired to convey is the fact that<br />
they are also an important component of our ecosystem<br />
and that is the way they are going to be come what may.<br />
Practically every animal and plant species is<br />
parasitized by one or more species of parasites. Man<br />
alone is parasitized by some 36 different species of<br />
nematodes. Add to this list, the species of flukes,<br />
tapeworms, insects and the microbial organisms <strong>for</strong><br />
which we the human beings are the most favored host.<br />
Nobody would dispute that in recent times man has<br />
caused great destruction to the world ecology. An<br />
exponential rise in human population has also<br />
contributed to a great extent to environmental<br />
degradation and in the rise of pollution levels the<br />
world-over particularly in the Third World countries.<br />
Many of the parasitic diseases that had declined or<br />
almost disappeared in the past such as Malaria have<br />
come back with a vengeance. We need to evolve global<br />
strategies to deal with parasitic diseases as the<br />
parasites do not respect man-made national<br />
boundaries. <strong>The</strong> first step in this direction would be the<br />
environmental management of parasites and to make<br />
ef<strong>for</strong>ts to determine the weakest points in their life<br />
cycles which could be exploited to tackle them more<br />
effectively. <strong>The</strong> world-wide ef<strong>for</strong>ts to develop vaccine<br />
against malarial parasite are indeed laudable but we<br />
must also direct our ef<strong>for</strong>ts more vigorously towards<br />
breaking the life cycle of mosquitoes in their aquatic<br />
phase of life. This would be far easier and cheaper and<br />
simultaneously it may also help in combating other<br />
mosquito-transmitted diseases. Though multi-<br />
pronged ef<strong>for</strong>ts would also prove effective <strong>for</strong> certain<br />
parasites but the cost-benefit factor should not be<br />
ignored be<strong>for</strong>e embarking on such a strategy. <strong>The</strong><br />
Developed World has already succeeded to a great<br />
extent in combating parasitic diseases in their<br />
their back the females migrate to this area rather than to<br />
the extremities.<br />
<strong>The</strong> mermithid nematodes produce very peculiar type<br />
of eggs that have long filamentous branches. <strong>The</strong>se get<br />
entangled to the plants so as to hold the eggs. <strong>The</strong><br />
grasshoppers /locusts become infected while eating<br />
grasses and foliage containing the eggs. Similarly, the<br />
larval cercariae of Fasciola hepatica, the liver fluke<br />
encyst on grasses and other type of vegetation waiting<br />
to be eaten and ingested by sheep or other definitive<br />
hosts. <strong>The</strong> infective juveniles of Agamermis<br />
decaudata emerge from the soil cavities and climb<br />
nearby vegetation especially during periods of high<br />
moisture such as dew or rain. <strong>The</strong>re they wait <strong>for</strong> the<br />
newly hatched insect nymphs and penetrate their<br />
hemocoel through the body wall as and when they<br />
come in their contact. One could go on like this quoting<br />
innumerable interesting example of host-parasite<br />
interactions with particular reference to their mutual<br />
compatibility.<br />
All parasitic associations initially begin only as a<br />
matter of chance contact between two species of<br />
organisms. None of these is a parasite to start with,<br />
though one of them happens to be smaller and weaker<br />
then the other one. <strong>The</strong> sole intention of the weaker<br />
organism is to feed on the larger one, ecto- or<br />
endoparasitically. <strong>The</strong> future generations of these two<br />
species maintain this interaction and thus a new<br />
parasite-host association begins. During the initial<br />
period of interaction, the parasite will still have the<br />
ability to survive on its own in the absence of its host<br />
because it is only in the facultative and not the obligate<br />
parasitic stage. But in due course of evolution and<br />
struggle <strong>for</strong> survival, the parasite also gradually<br />
becomes more and more dependent on the host.<br />
Ultimately, a stage reaches when the parasite can not<br />
survive without the host. At this point, the parasite has<br />
already become obligatory in nature and it is now<br />
entirely dependent upon its host. While the parasite is<br />
gradually in the process of evolving from the<br />
facultative to the obligatory stage, the host simply does<br />
not remain a silent spectator. Simultaneously, it also<br />
makes ef<strong>for</strong>ts using its tissue reactions and the<br />
mechanism of resistance or immunity to fight off the<br />
parasitic invasion thereby minimising the damage to
4 Jairajpuri : Presidential Address<br />
JPD : Vol. 28 (1), 2004<br />
countries. But the people in the Developing World and purposes. Further, a lot of man-hours are lost annually<br />
more so in the Underdeveloped World are grappling due to the health problems of working population<br />
with these health problems in a big way not only <strong>for</strong> which could otherwise have been gainfully utilized.<br />
man himself but also <strong>for</strong> his live-stock, the agricultural This is beside the yearly deaths that are caused due to<br />
and horticultural crops. Huge sums of money is spent parasitic diseases. It should be our endeavor to free<br />
every year from the already depleted resources of these humanity from the ill effects of diseases due to<br />
countries in fighting off parasitic and other diseases parasites. May this Global Meet turn out to be an<br />
which could have been spent <strong>for</strong> development important step in this direction !
Journal of Parasitic Diseases<br />
Vol. 28 (1) June 2004, pp. 5-16<br />
Review<br />
Role of DNA Microarrary Technology <strong>for</strong> understanding<br />
differential gene expression in Parasitic Diseases<br />
1 1<br />
ANJAN DEBNATH , ABHIK SEN, JAMES H. MCKERROW AND PRADEEP DAS*<br />
National Institute of Cholera & Enteric Diseases, P-33, C.I.T Road, Scheme-XM, Kolkata-700010, India<br />
1,<br />
and Sandler Center <strong>for</strong> Basic Research in Parasitic disease University of Cali<strong>for</strong>nia,<br />
San Francisco, CA 94143, USA<br />
DNA microarray holds exceptional promise as a technique to exploit the explosion of genomic in<strong>for</strong>mation.<br />
It is, in principle and practice, an extension of hybridization-based methods that have been used to identify<br />
and quantitate nucleic acids in biological samples. But unlike other techniques, it allows <strong>for</strong> tens of<br />
thousands of genes to be represented in areas smaller than standard glass microscopic slides called 'chips'.<br />
Samples of interest are fluorescently labeled and allowed to hybridize to the array; after hybridization<br />
followed by washing steps, an image of the array is acquired and the representation of individual nucleic acid<br />
species in the sample is reflected by the amount of hybridization to complementary DNAs immobilized in<br />
known positions on the array. Microarrays are typically constructed based on either sequence in<strong>for</strong>mation<br />
obtained from genome sequencing projects or by arraying inserts from a random fragment genomic library.<br />
<strong>The</strong> technique is now widely used in understanding the host response as well as identification of pathogen<br />
virulence determinants. It has also been successfully employed in the study of differential gene expression in<br />
disease and health, high-throughput detection of single-nucleotide polymorphisms, genome mapping,<br />
mutation screening; and identification of potential vaccine targets. Among the parasites, microarray<br />
analysis has been recently applied to study various aspects ,of the pathogenesis and life cycles of Plasmodium<br />
falciparum, Toxoplasma gondii, Leishmania spp., Cryptosporidium parvum, Trypanosoma cruzi,<br />
Trypanosoma brucei, Entamoeba histolytica, Schistosoma mansoni, Ascaris suum and Trichostrongylus<br />
vitrinus. To illustrate the utility of this technique in gene expression studies some of the results of E.<br />
histolytica and human collagen interaction will be discussed.<br />
Keywords: Array, Collagen, Entamoeba histolytica, Genome, Parasites<br />
P<br />
arasitic diseases have a debilitating impact on<br />
human health worldwide, particularly in<br />
developing countries. Malaria, Schistosomiasis,<br />
Leishmaniasis and Trypanosomiasis still result in a<br />
large number of deaths each year, while drug<br />
resistance in malaria is increasing as a public health<br />
problem (Ellis et al 2003). Among the gut-dwelling<br />
parasitic protozoa, Entamoeba histolytica and Giardia<br />
intestinalis are frequent causes of acute diarrhoea<br />
while intracellular parasite Cryptosporidium parvum<br />
is commonly associated with diarrhoea in infants. In<br />
agriculture, the steadfast presence of gastrointestinal<br />
* Corresponding Author<br />
parasites in livestock throughout the world is<br />
responsible <strong>for</strong> significant economic loss. Control<br />
measures against gastrointestinal nematodes alone<br />
cost over 1 billion pound annually (McDonald, 2003)<br />
but resistance in parasitic worms is increasing at such<br />
an alarming rate that many drugs will soon be rendered<br />
useless to the livestock industry.<br />
In order to identify new genes with key cellular<br />
functions, targets <strong>for</strong> new drugs, and new antigens<br />
which could be useful in the diagnostic or <strong>for</strong> vaccine<br />
development, scientists from developing and<br />
developed countries planned and initiated a number of<br />
parasite genome projects. Thus, the genomes of E.<br />
histolytica, Giardia lamblia, Plasmodium falciparum,<br />
Schistosoma mansoni, Trypanosoma cruzi,
6 DNA Microarray in parasitic diseases<br />
JPD : Vol. 28 (1), 2004<br />
Leishmania major, \Trypanosoma brucei, Brugia virulence determinants that enable E. histolytica to<br />
malayi and other pathogenic nematodes, are now cause invasive colonic and hepatic disease have not<br />
under study (Degrave et al 2001). Access to all open been identified and the genetic factors that make E.<br />
reading frames from the genome of these organisms is histolytica virulent and E. dispar avirulent remain to<br />
imminent, and a fully annotated genome of parasites be elucidated. With the advent of microarray technique<br />
will soon be available. Sequence function in itself has our understanding of the complex genetic processes<br />
already become too complex to be dissected by the underlying the interaction between Entamoeba and the<br />
classical reductionist approach to molecular biology, host will be accelerated and consequently, will provide<br />
where genes are analyzed individually (Rathod et al valuable insight into design of novel means to prevent,<br />
2002). Moreover, DNA sequences alone tell us little treat and diagnose infection.<br />
about the dynamic processes within the parasite.<br />
<strong>The</strong>re<strong>for</strong>e, the task facing parasitologists is to extract<br />
WHAT ARE DNA MICROARRAYS?<br />
useful in<strong>for</strong>mation from rapidly accumulating genome A microarray is a high-density microscopic<br />
data (Ashton et al 2001). Functional genomics has arrangement of nucleic acid targets immobilized on a<br />
wider application in identifying potential protein solid substrate. Samples of interest are fluorescently<br />
targets <strong>for</strong> vaccination or novel drug targeting. Global labelled and allowed to hybridize to the array.<br />
profiling of gene expression is one attractive approach Following hybridization an image of the array is<br />
to assessing function. Because a gene is usually acquired and the representation of individual nucleic<br />
transcribed only when and where its function is acid species in the sample is reflected by the amount of<br />
required, determining the locations and conditions hybridization to complementary DNAs immobilized<br />
under which a gene is expressed allows inferences in known positions on the array (Eisen and Brown,<br />
about its function. Several independent high- 1999).<br />
throughput methods, such as serial analysis of gene<br />
expression (SAGE), differential display and expressed MICROARRAY FABRICATION<br />
sequence tag (EST) analysis, are available <strong>for</strong> gene<br />
Microarraying of double-stranded or single-stranded<br />
expression studies and may enable function annotation<br />
DNA fragments on a glass microscopic slide is being<br />
of sequenced genomes. But DNA microarray<br />
used extensively owing to the accessibility, cost<br />
technology stands out <strong>for</strong> its simplicity,<br />
effectiveness and proven success of this approach.,<br />
comprehensiveness, data consistency, and high Four broad variations of DNA microarray are now<br />
throughput (Cummings and Relman, 2000). <strong>The</strong><br />
being constructed in parasite laboratories worldwide:<br />
purpose of this review is to focus on the general<br />
methodology <strong>for</strong> microarray development with special Random (shotgun) microarrays<br />
emphasis on a recently developed E. histolytica<br />
<strong>The</strong>se have been constructed either out of a mungmicroarray<br />
highlighting the applications of<br />
bean-nuclease digested genomic DNA (gDNA) library<br />
microarray in understanding the molecular basis of<br />
or from a randomly sheared (using nebulizer) gDNA<br />
host-pathogen interaction of different parasites.<br />
library. In most slides, duplicate spots are attached as<br />
Amoebiasis, caused by the protozoan parasite E. controls, either next to or distant from the first spot,<br />
histolytica continues to be the major cause of with up to 20,000 spots per slide. In gene-expression<br />
morbidity and mortality. WHO in its most recent experiments, mRNA samples are converted to cDNA<br />
estimates has placed the death toll from amoebiasis at and are fluorescently labelled with flurochrome dyes,<br />
40,000-100,000 lives annually (WHO, 1997). Cy5 (red) and Cy3 (green), either during reverse<br />
Significant progress has been made in understanding transcription or afterwards. <strong>The</strong> two samples are then<br />
the mechanisms that make these intestinal protozoa incubated with the microarray slide and the<br />
unique in its ability to cause invasive disease in fluorescently labelled probe DNA hybridizes to the<br />
humans. Also, the genetic mysteries of the organisms immobilized target DNA with high sensitivity and<br />
are beginning to be revealed; helping to explain the specificity. <strong>The</strong> slide is scanned and the intensity of red<br />
pathogenic difference versus closely related and green fluorescence at each spot is proportional to<br />
commensal organisms such as Entamoeba dispar the amount of Cy5 and Cy3-labelled cDNA<br />
(Stauffer and Ravdin, 2003). But still the majority of respectively that has hybridized to the DNA at that
JPD : Vol. 28 (1), 2004<br />
DNA Microarray in parasitic diseases<br />
7<br />
spot. This quantitative ratio between the two DEVELOPMENT OF E. histolytica SHOTGUN<br />
fluorescent dyes gives in<strong>for</strong>mation about the MICROARRAY<br />
comparative over expression or under expression of<br />
genes in an experimental sample. <strong>The</strong> advantage of<br />
As the genome of E. histolytica remains to be fully<br />
competitive hybridization is that the ratio remains the<br />
sequenced, it is currently not possible to construct<br />
same even if there is differences in the amount of target<br />
gene-specific microarrays representing complete E.<br />
DNA present (Bryant et al 2004). Since the<br />
histolytica genome. However, given the relatively<br />
sequences of the arrayed gDNA fragments on a<br />
small size of the E. histolytica genome, valuable<br />
shotgun microarray are unknown, it necessitates<br />
in<strong>for</strong>mation on gene expression related to<br />
post-hybridization sequencing of interesting<br />
environmental signals can be initially assessed by<br />
clones. Shotgun gDNA microarrays offer the<br />
microarray analyses. <strong>The</strong> E. histolytica genome has<br />
possibility of an immediate, genome-scale short intergenic regions and relatively few and short<br />
experiment even in the absence of a complete<br />
introns. This makes the use of genomic library <strong>for</strong><br />
genome sequence.<br />
microarray development a feasible approach (Singh<br />
and Shah, 2002). Random shotgun collections provide<br />
Gene-specific microarrays<br />
an efficient means <strong>for</strong> generating DNAs capable of<br />
identifying large fraction of Entamoeba mRNAs, and<br />
<strong>The</strong>se are being developed from large sets of wellare<br />
there<strong>for</strong>e well suited <strong>for</strong> expression profiling<br />
characterized gene sequence tag (GST) and EST<br />
studies. It is relatively inexpensive to prepare DNA<br />
libraries. Such arrays have the advantage of starting<br />
collections, and expression-profiling analysis need<br />
with known sequences. <strong>The</strong> representation of genomic<br />
not wait <strong>for</strong> the availability of complete genomic DNA<br />
sequences could be biased in EST sequences because<br />
sequence (Akopyants et al 2001). Due to the random<br />
the library is usually derived from a specific stage of<br />
nature of the shotgun cloning procedure, there is an<br />
parasite life cycle. Nonetheless, an enormous amount<br />
unbiased representation of sequences in the genomic<br />
of fresh in<strong>for</strong>mation can be derived from such arrays<br />
library, so genes that are rarely transcribed have the<br />
(Rathod et al 2002).<br />
same representation as highly expressed ones. Overall<br />
Double-stranded ordered arrays<br />
genome representation is usually good in shotgun<br />
libraries with comparatively little variation across the<br />
Following publication of full-length sequences of genome.Thus, even if randomly selected clone inserts<br />
different chromosomes, a physical map of annotated are used as probes, there should be good coverage and<br />
genes in a specific linear order is now available. relatively little redundancy. Also, not only coding but<br />
Amplified genes from genome template using also intergenic regions can be studied (Diehl et al 2002).<br />
sequence-specific primers are being arrayed alongside<br />
other gDNA fragments to provide additional To begin to identify global changes in E. histolytica<br />
insights into transcriptional regulation mechanisms gene expression related to different stimuli, we<br />
(Rathod et al 2002),<br />
produced an E. histolytica microarray using a<br />
variation of the standard array technology.<br />
Long oligonucleotide array<br />
Approximately 1 kb inserts from an E. histolytica<br />
Long oligonucleotide can be spotted directly on slides.<br />
random fragment genomic library were isolated to<br />
Over the entire slide there are several different<br />
generate 'shotgun' microarrays (Debnath et al 2004).<br />
oligonucleotide sequences from the same gene. This Array construction<br />
range produces more robust results because the same<br />
gene can be probed independently several times in the Genomic DNA was sheared using the nebulizer<br />
same experiment. This approach is simpler because it (Invitrogen, USA) connected to the laboratory<br />
bypasses the laborious PCR, DNA purification and gel compressed air line. <strong>The</strong> sheared DNA was blunt-end<br />
analysis steps of traditional array construction, and the repaired with T4 DNA and Klenow polymerases, and<br />
exact sequence of the 70mer DNA strand are known <strong>for</strong> the ends were dephosphorylated with calf intestinal<br />
®<br />
each spot (Rathod et al 2002). It also allows the user to phosphatase to create a suitable substrate <strong>for</strong> TOPO<br />
avoid genes that are repetitive or homologous to other cloning (Invitrogen). DNA fragments were ligated into<br />
® ®<br />
known genes.<br />
pCR 4Blunt-TOPO vector (Invitrogen) and the
8 DNA Microarray in parasitic diseases<br />
JPD : Vol. 28 (1), 2004<br />
cloning reaction was electroporated into One Shot®<br />
TOP 10 Electrocomp E. coli (Invitrogen) and the<br />
trans<strong>for</strong>mants were incubated overnight on agar plates<br />
in the presence of X-gal and 100µg/ml of ampicillin<br />
(Debnath et al 2004).<br />
washing purpose (Holloway et al 2003c). Five<br />
microliter PCR products from each 96 well plate are<br />
transferred to 384-well printing plates (Genetix, USA)<br />
using a BioMek FX 96 well pipetting robot (Beckman<br />
Coulter, USA) and dried down in speed-vac (Savant,<br />
USA).<br />
Perhaps the most laborious aspect of generating<br />
spotted DNA microarrays is the production of In order to resuspend the array-ready material be<strong>for</strong>e<br />
concentrated solutions of array-ready DNA fragments printing, the optimal solvent <strong>for</strong> the DNA depends on<br />
by high-throughput PCR. <strong>The</strong> goals are to amplify the geometry of printing tip, the printing substrate<br />
inserts from a large percentage of clones, to avoid being used, the length of time the plates are exposed<br />
cross-contamination, and to purify the fragments from during printing, and the degree and type of<br />
the enzymes, nucleotides, primers, and other environmental control available in the printing device.<br />
components of PCR. To maximize the yield from a Moreover, the volume of solution in the wells of the<br />
single round of PCR amplifications and to enable the printing plates is important because overly full wells<br />
printing of thousands of microarray slides, PCRs are can lead to excessive wetting of the exterior surface of<br />
carried out in a volume of 100µl <strong>for</strong> each gene or pins and poor spot morphology as well as dripping.<br />
microan-ay element. <strong>The</strong>. yield of PCR is significantly Considering all these factors, 4 (µl of 3X SSC was<br />
improved by use of fresh cultures as a source of found to be the best solvent <strong>for</strong> DNA printing and also<br />
template DNA (Holloway etal 2003a).<br />
the required minimum volume to obtain consistent<br />
filling of the printing pins. DNA concentrations in the<br />
DNA yield, the presence of free primers or primer printing solution are typically in the range of 100-250<br />
dimers, and the number of bands in each reaction are<br />
µg/ml because there is a loss of sensitivity and an<br />
important measures of the quality of PCR-amplified<br />
material available <strong>for</strong> printing. Quality control is<br />
per<strong>for</strong>med prior to or after purification of PCR<br />
products. Analysis be<strong>for</strong>e purification avoids the cost<br />
and ef<strong>for</strong>t involved in processing PCRs (Holloway et<br />
al 2003b). Commercially available gel systems (E-<br />
Gel® 96 gel, Invitrogen) provide rapid efficient<br />
analysis. PCR products are assayed via 96 well agarose<br />
gel electrophoresis. Although in some cases PCR<br />
products may contain an extra band, a single<br />
prominent PCR product is visible and the extra band<br />
does not interfere with array hybridization.<br />
underestimation of the degree of differential<br />
expression between samples if the DNA concentration<br />
is lower than 100µg/ml. Also, excessively<br />
concentrated solutions waste valuable material and<br />
can lead to smearing or comet tails in hybridization<br />
(Holloway et al 2003d).<br />
<strong>The</strong> purification of PCR products be<strong>for</strong>e the printing<br />
of the microarray serves to remove enzymes or other<br />
proteins that can clog printing tips, oligonucleotide<br />
primers that can compete with the product <strong>for</strong> substrate<br />
binding, and salts that could affect binding to the<br />
substrate. To clean up PCR products and prepare the<br />
DNA <strong>for</strong> printing, the PCR products are isopropanol-<br />
precipitated. During precipitation isopropanol is used<br />
instead of ethaaol to reduce the volume of the<br />
precipitation mixture to an amount that will fit in the<br />
tissue culture plates. Since some batches of absolute<br />
ethanol (100%) had been reported to contain<br />
fluorescent contaminants that generate high<br />
backgrounds in microarray experiments, so 70%<br />
ethanol is prepared from 95% ethanol <strong>for</strong> DNA<br />
Although arrays of DNAs on porous membranes have<br />
been in use <strong>for</strong> years to per<strong>for</strong>m parallel hybridization<br />
studies, technical advances made it possible to<br />
trans<strong>for</strong>m these rather clumsy membranes into much<br />
more useful and efficient methods to generate arrays<br />
with high densities of DNAs, allowing <strong>for</strong> tens of<br />
thousands of genes to be represented in areas smaller<br />
than standard glass microscopic slides. Glass slides<br />
have been a favoured solid support <strong>for</strong> immobilization<br />
ofDNAs because of their easy availability, low<br />
intrinsic fluorescence, high transparency, good<br />
thermal properties, excellent rigidity, and<br />
straight<strong>for</strong>ward chemistries <strong>for</strong> modification of the<br />
surface. Due to the nonporous nature of glass, the<br />
labelled cDNAs have direct access to immobilized<br />
DNAs without limitations of internal diffusion<br />
(Holloway et al 2002).<br />
<strong>The</strong> best choice <strong>for</strong> coating glass microscopic slides is<br />
poly-L-lysine. Many alternate coating materials had<br />
been tried, including numerous silanes, but none ating
JPD : Vol. 28 (1), 2004<br />
DNA Microarray in parasitic diseases<br />
9<br />
proved to be as simple and reliable as polylysine. It is first few printed spots, giving rise to large-irregular<br />
not only inexpensive, but also readily accessible and spots (Childs et al 2003). DNA samples in 3X SSC in<br />
generally provides good results. <strong>The</strong> coating attaches 384-well microtiter plates are placed in the plate<br />
firmly to the slide and binds tightly to DNA spotted holder. <strong>The</strong> robot positions a cluster of specialized<br />
onto the surface. <strong>The</strong> binding of DNA to poly-L-lysine spring-loaded 16 or 32 printing tips into adjacent wells<br />
is complex but essentially involves charge interactions of the DNA source plates, to a depth of about 1 mm,<br />
(Childs et al 2003). Although most slides come in filling the reservoir slot of each tip with approximately<br />
boxes labelled "precleaned", it is essential that they 1µ<br />
l of DNA solution. <strong>The</strong> tips are then lightly tapped at<br />
should be stripped clean prior to coating with alkaline identical positions on each slide, leaving a small (
10 DNA Microarray in parasitic diseases<br />
JPD : Vol. 28 (1), 2004<br />
between print runs, the printing materials are a free carboxylate group on the other end of the chain. In<br />
completely dried in a vacuum concentrator at the end addition to converting the amino groups of the lysines to<br />
of each run <strong>for</strong> storing. At the end of the print run, the amides, this new moiety creates a negatively charged<br />
slides are allowed to dry to facilitate binding of the surface that further reduces nonspecific binding of<br />
printed DNA and the slide coating.<br />
DNA. As the blocking reagent has limited stability in<br />
solution, it is important to carry out the process very<br />
<strong>The</strong> E. histolytica array was constructed by printing<br />
quickly. <strong>The</strong> last step during microarray post-<br />
9600 PCR-amplified inserts from twenty five 384-well<br />
processing consists of boiling the arrays. <strong>The</strong> boiling<br />
plates using the arraying robot with a similarly sized<br />
step is meant to denature the double stranded DNA<br />
print-tip cluster of 32 tips spaced <strong>for</strong> 384-well plates.<br />
molecules and thus enhances hybridization<br />
<strong>The</strong> size of the genome of E. histolytica is predicted to<br />
availability(Eisen and Brown, 1999,<br />
be 20 Mb. <strong>The</strong> average size of the insert applied to the<br />
http://www.microarrays.org/pdfs/PostProcessing2001<br />
array was 1 kb and the microarray was constructed by<br />
.pdf).<br />
printing 9600 PCR-amplified inserts from E. histolytica<br />
genomic DNA library. <strong>The</strong>oretically, the microarray Use of E. histolytica microarray to identify genomic<br />
covered 9.6 Mb or almost half of E. histolytica genome. responses by Entamoeba to collagen binding<br />
Since the E. histolytica genome has large number of<br />
repetitive sequences, some inserts will have identical or<br />
Since collagen is a major component of the epithelial<br />
nearly identical sequences. Based on these calculations,<br />
basal lamina, and the extracellular matrix of the<br />
we estimate that this microarray may cover one half to<br />
intestine, E. histolytica encounters collagen during<br />
one-third of the E. histolytica genome (Debnath et al<br />
colonic invasion. In vitro, incubation of trophozoites<br />
2004).<br />
with collagen type I leads to parasite activation which<br />
includes the release of electron-dense granules<br />
After a complete set of samples is spotted, slides were (EDGs), containing factors considered important in<br />
processed <strong>for</strong> hybridization, checked once again, to the pathogenicity of this parasite (Martinez-Palomo et<br />
ensure that spotted DNA attaches to the slide stably. al 1987; Munoz et al 1991, 1996; Serrano et al 1996).<br />
Active groups on the slide surface are deactivated, Interaction of trophozoites with human collagen type I<br />
unbound DNA is removed and the target DNA is triggers biochemical cascades (Perez et al 1996), but<br />
denatured <strong>for</strong> hybridization. Three steps are required to the mechanism of activation and the complement of<br />
prepare printed arrays <strong>for</strong> hybridization: rehydration downstream products released has not been<br />
and snap drying, blocking, and denaturation. Since the elucidated. To identify cellular mechanisms of<br />
DNA solution dried to the outer edge of the spot during trophozoite activation, we assessed global variations<br />
the printing process, the goal of rehydration is to allow in gene - expression during collagen interaction with<br />
the DNA a chance to more evenly distribute across the E. histolytica. <strong>The</strong> arrays were probed with<br />
surface of the spot. This helps to eliminate the donut- differentially labelled cDNAs prepared from total<br />
shaped appearance of the spots and increases the RNA isolated from collagen-activated and nonamount<br />
of total DNA bound after processing. <strong>The</strong> activated cells (Debnath et al 2004).<br />
rehydration is a delicate process, as insufficient<br />
rehydration can produce irregular spot sizes and can Typically, fluorescently labelled cDNA is generated<br />
reduce overall hybridization intensity, while excessive<br />
by incorporation of dye-conjugated nucleotide<br />
rehydration can cause spots to fuse. Arrays on poly-Lthese<br />
fluorescent nucleotide analogs are quite<br />
analogs during the reverse transcription process. But<br />
lysine coated slides require blocking of exposed amines<br />
prior to hybridization to prevent binding of labelled<br />
expensive and they have low efficiency of<br />
material indiscriminately and nonspecifically to the<br />
incorporation by polymerase. Linkage of a primary<br />
surface. <strong>The</strong> blocking process, which is the most critical<br />
amine to a N-hydroxysuccinimidyl ester group<br />
step in post processing, is a competition between excess<br />
attached to a dye (Cy3 and Cy5) provides an<br />
DNA molecules leaving spots to bind near-by exposed<br />
alternative probe labelling method that increases the<br />
lysine, and the succinic anhydride used <strong>for</strong> blocking<br />
fluorescent intensity of the final product and decreases<br />
reacts and caps the amines. <strong>The</strong> charged amino group of<br />
the cost. <strong>The</strong> reactive amine derivative ofdUTP, 5-(3-<br />
the lysine carries out a nucleophilic attack on one of the<br />
aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-<br />
carboxyl carbons, <strong>for</strong>ming an amide bond and exposing<br />
dUTP), is used during labelling because it can be
JPD : Vol. 28 (1), 2004<br />
DNA Microarray in parasitic diseases<br />
11<br />
incorporated by a variety of RNA-dependent and assign coordinates to each of the spots. <strong>The</strong> laser<br />
DNA-dependent DNA polymerases. After removing scanner produces light with a wavelength appropriate<br />
free nucleotides, the amino-allyl labelled samples can <strong>for</strong> the excitation spectra of the dyes used (<strong>for</strong> Cy3 this<br />
be coupled to dye, purified again, and then applied to is green light around 540 nm; <strong>for</strong> Cy5 red light around<br />
the microarray. For the labelling of a complex mixture 650 nm) (Eisen and Brown, 1999). To extract<br />
of samples, as is the case with mRNA, the labelling meaningful data and also to draw logical and<br />
density can be roughly controlled by limiting the reproducible conclusions from the massive amount of<br />
amount of aa-dUTP in relation to the amount of dTTP. in<strong>for</strong>mation distilled from the complex E. histolytica<br />
This choice depends on the overall A/T content of the DNA chip data, effective tools <strong>for</strong> data mining are<br />
genome in question. A ratio of 3 aa-dUTPs to 2 dTTPs critical. <strong>The</strong> data analysis was per<strong>for</strong>med using<br />
worked good <strong>for</strong> E. histolytica genomic DNA arrays. powerful analytical GenePix Pro 4.0.1.9 software.<br />
Compared to enzymatic incorporation of dye, the Local background was subtracted from the value of<br />
procedure <strong>for</strong> post-coupling of reactive dyes is a each spot on the array. Only spots in which almost<br />
longer process both in terms.of time and steps. more than 70% of the pixels had a signal above<br />
However, the added advantage of brightness, lack of background of at least twice the standard deviation<br />
enzymatic bias, and low cost is likely to be worth the (S.D.) of the local background were considered in<br />
extra ef<strong>for</strong>t (DeRisi, 2003). cDNA from non-activated subsequent data analysis. <strong>The</strong> data were normalized<br />
E. histolytica was coupled with Cy3 dye and cDNA using the NOMAD microarray database.<br />
from collagen-activated E. histolytica was coupled Normalization of expression data is per<strong>for</strong>med to<br />
with Cy5 dye.<br />
adjust the individual hybridization intensities and to<br />
balance them appropriately so that meaningful<br />
During hybridization, buffer of high ionic strength is<br />
biological comparisons can be made. It minimizes<br />
used to reduce electrostatic repulsion and to promote<br />
unequal quantities of starting RNA, differences in<br />
association of complementary strands. Detergent is<br />
labelling or detection efficiencies between the<br />
used to minimize background noise. <strong>The</strong> hybridization<br />
fluorescent dyes used, and systematic biases in the<br />
mixture is heated to reduce the background (Ideker el<br />
measured expression levels (Quackenbush, 2002).<br />
al 2003). <strong>The</strong> probe is carefully pipetted onto the<br />
<strong>The</strong> Cy5/Cy3 fluorescence ratios and logiocenter<br />
of the array and a coverslip is placed in such a<br />
trans<strong>for</strong>med ratios were calculated from the<br />
way so that <strong>for</strong>mation of bubbles could be avoided<br />
normalized values. In order to gain speciflcity and the<br />
since air bubbles and inhomogeneities in the flatness<br />
threshold expression level of gene that can be<br />
of the slide and/or coverslip could cause locally<br />
considered significant, the gene expression profile <strong>for</strong><br />
decreased signal. To provide humidity in the<br />
each individual gene is established (Dhiman et al<br />
hybridization chamber and also to ensure that the<br />
2001). <strong>The</strong> interesting genes are visually identified<br />
probe mixture does not dehydrate during<br />
using a scatterplot of the data. In general, a factor of<br />
hybridization, a larger volume of 3X SSC is added on a<br />
two change in expression is considered to be adequate<br />
separate part of the slide and the chambers are well<br />
to decide whether a particular gene is upregulated<br />
sealed. This decreases high background often<br />
(Groen, 2001). <strong>The</strong>re<strong>for</strong>e, genes whose expression<br />
observed near the edges of the coverslip (Eisen and<br />
changed 2-fold or more relative to collagen-uninduced<br />
Brown, 1999). Although optimal time <strong>for</strong><br />
E. histolytica were considered positive and selected<br />
hybridization depends on the complexity and<br />
<strong>for</strong> further sequencing experiments.<br />
concentration of the sample, 16 hour hybridization<br />
provided good result. Both hybridization and washing Cluster analysis is used to group together genes with<br />
are carried out under conditions of moderate to high similar patterns of expression. Although various<br />
stringency to suppress cross-hybridization.<br />
clustering methods can usefully organize gene<br />
expression measurements, the resulting ordered but<br />
After hybridization, a fluorescent image is acquired<br />
still massive collection of numbers remains difficult to<br />
<strong>for</strong> both the fluorescent dyes used by a scanner<br />
assimilate. <strong>The</strong> end product is a representation of<br />
(Figure1). <strong>The</strong> photomultiplier tube (PMT) voltage is<br />
complex gene expression data that, through statistical<br />
set in such a way so that brightest pixels are just below<br />
organization and graphical display, allows to<br />
the level of saturation. Increased PMT setting can<br />
assimilate and explore the data in a natural intuitive<br />
increase both signal and noise. Gridding helps to
12 DNA Microarray in parasitic diseases<br />
JPD : Vol. 28 (1), 2004<br />
<strong>The</strong> faithfulness of the shotgun DNA microarray <strong>for</strong><br />
reporting collagen-specific gene expression was<br />
apparent from the fact that due to the size selection<br />
process prior to the shotgun cloning, the fragments<br />
were almost similar in length. Also, variation in the<br />
efficiency of priming events was minimized, since an<br />
identical primer pair was used in all reactions. Three<br />
separate hybridizations from three independent cDNA<br />
preparations showed virtually identical differential<br />
hybridization patterns and in total, 225 clones out of<br />
9600 white colonies printed on the DNA array were<br />
actually upregulated (= or > 2) due to collagen<br />
activation. Clustering the genes enhanced their<br />
similarities and made it easy to correlate those that<br />
might have a relationship with the specific event.<br />
Sequencing results of initial 14 clones showed good<br />
homology with functionally important proteins. Few<br />
genes that were shown by the DNA microarray to be<br />
manner. To illustrate this approach, pairwise average-<br />
linkage cluster analysis is applied to collected gene<br />
expression data (Figure 2). This method is a <strong>for</strong>m of<br />
hierarchical clustering. <strong>The</strong> computed trees are used to<br />
order genes in the original data table, so that genes or<br />
groups of genes with similar expression patterns are<br />
adjacent. <strong>The</strong> ordered table then is displayed<br />
graphically with a representation of the tree to indicate<br />
the relationships among genes (Eisen et al 1998).<br />
over expressed during collagen interaction were<br />
validated by RT-PCR, affirming the reliability of this<br />
microarray (Debnath et al 2004).<strong>The</strong> homology<br />
analysis of the sequences of 14 collagen activated over<br />
expressed cDNA fragments corresponded with SH3<br />
domain-containing protein SH3P17 (accession no.<br />
AAC50592), intersectin 1 (accession no. 042287),<br />
VAV (Homo sapiens) (accession no. CAA34383),<br />
growth factor receptor-bound protein 2 (GRB2 adapter<br />
protein) (SH2/SH3 adapter GRB2) (ASH protein)<br />
(accession no. Q07883), unconventional myosin IB<br />
(E. histolytica) (accession no. AAC47535);<br />
asparaginyl-tRNA synthetase (Mus musculus)<br />
(accession no. XP_128957); cysteine proteinase 1 and<br />
2 precursors (accession no.s Q01957, Q01958); E.<br />
histolytica gene <strong>for</strong> pore-<strong>for</strong>ming peptide (accession<br />
no. X70851); E. histolytica putative GTPase and<br />
putative RNA binding protein genes, partial cds; and<br />
ehapt2 retrotransponson (accession no.s AY141200,<br />
AY141201); 40S ribosomal protein S24 and ribosomal<br />
Fig. 1. E. histolytica genomic microarray of 6144 clones.<br />
Collagen-induced mRNA expression profile was monitored by<br />
microarray hybridization analysis. A fluorescently labelled (Cy3)<br />
cDNA probe was prepared from total RNA isolated from normal E<br />
histolytica trophozoites by reverse transcription and a Cy5-<br />
labelled second probe was prepared from equal quantity of total<br />
RNA isolated from collagen-activated E. histolytica trophozoites<br />
by reverse transcription. In this image, hybridization of Cy3-<br />
labelled cDNA is represented as a green signal and hybridization<br />
of Cy5-labelled cDNA is represented as a red signal. Thus, genes<br />
induced or repressed due to interaction of amoebae with collagen<br />
appear as red and green spots, respectively. Genes expressed at<br />
roughly equal levels appear as yellow spots.<br />
Fig. 2. A representative gene expression map from a portion of<br />
whole microarray hybridization. Descriptions of each experiment<br />
appear above each column (EhCSlpl-056, EhCSlp2-055 and<br />
EhCSlp2-060), whereas gene identifiers appear to the right of each<br />
row. Red cells indicate increased expression due to collagen<br />
compared to non-activated trophozoites; green cells indicate<br />
decreased expression; grey cells represent negative or undetectable<br />
expression. <strong>The</strong> intensity of the colour corresponds to the<br />
magnitude of change with brighter colours showing larger<br />
differences versus "non-activated trophozoites.
JPD : Vol. 28 (1), 2004<br />
DNA Microarray in parasitic diseases<br />
13<br />
protein S19 (accession no. NP_187143); E. histolytica more sophisticated Plasmodium microarray spotted<br />
putative mitochondrial type and endoplasmic with open-reading frame-specific oligonucleotides<br />
reticulum-like HSP70 gene (accession no.s showed a unique coordinated expression of genes<br />
AY141198,AY141199).<br />
encoding ribosomal proteins, multiple factors <strong>for</strong><br />
transcription' and translation, enzymes of biosynthetic<br />
<strong>The</strong> probable functions of collagen-induced genes<br />
and catabolic pathways, or merozoite adherence and<br />
include coding <strong>for</strong> components of a signalling cascade<br />
invasion machinery (Bozdech et al 2003).<br />
previously hypothesized to transmit responses<br />
induced by cell attachment, adapter proteins <strong>for</strong> <strong>The</strong> application of microarray analysis to Toxoplasma<br />
vesicle <strong>for</strong>mation, proteins implicated in cytoskeletal gondii gene expression has advanced to the analysis of<br />
reorganization and locomotion, and tissue degradation coordinate regulation of cluster of genes. T gondii<br />
- cysteine proteinases and the amoebapore (Debnath et cDNA microarrays were used to study changes in<br />
al 2004).<br />
transcript levels during tachyzoite-to-bradyzoite<br />
differentiation (Cleary et al 2002). Cluster analysis<br />
<strong>The</strong> molecular consequences of collagen adherence on<br />
identified coordinate bradyzoite expression of clones<br />
E. histolytica provide confidence that many of the<br />
that encode cell-surface proteins and proteincollagen-responsive<br />
genes encode proteins that are<br />
modifying enzymes. <strong>The</strong> groups of genes that were<br />
relevant to the virulence of the parasite. <strong>The</strong> findings<br />
repressed in bradyzoites included several that encode<br />
also provide an insight into collagen-induced<br />
secretory organelle proteins and regulatory enzymes.<br />
signalling in amoebae, and strengthen the notion that a<br />
<strong>The</strong> same microarray, used to compare global gene<br />
genetic program is stimulated by interaction of E.<br />
expression in wild-type cell lines with chemically<br />
histolytica with extracellular matrix.<br />
induced, tachyzoite-to-bradyzoite differentiationdefective<br />
Taken together, the development of E. histolytica<br />
cell lines allowed the construction of a model<br />
microarray helped to identify global changes in E. of transcription regulation during bradyzoite<br />
histolytica gene expression related to collagen development (Singh et al 2002).<br />
activation, which in turn, provided a foundation <strong>for</strong> a<br />
A. C. parvum oligonucleotide microarray was<br />
more comprehensive analysis of Entamoeba<br />
designed to specifically detect Cryptosporidium spp.,<br />
behaviour.<br />
differentiate between the principle genotypes known<br />
MICROARRAY PROGRESS WITH OTHER to infect humans and demonstrated the potential to<br />
PROTOZOAN PARASITES<br />
differentiate between other closely related isolates and<br />
species through single-nucleotide mismatch<br />
As a tool to discover new genes and to identify discrimination (Straub et al 2002).<br />
coordinate expression of family of genes, the<br />
microarray has already proven effective <strong>for</strong> the In contrast to the apicomplexan microarray reports,<br />
apicomplexan parasites Plasmodium and Toxoplasma studies of kinetoplastids have not advanced<br />
(Duncan, 2004).<br />
significantly beyond validation of the data and<br />
discovery of new genes (Duncan, 2004). A genomic<br />
<strong>The</strong> earliest malaria microarrays were constructed microarray study of Trypanosoma brucei compared<br />
from 3648 random inserts from a P. falciparum mung gene expression in bloodstream and procyclic<br />
bean nuclease genomic library (Hayward et al 2000). trypanosomes. Hybridizations identified<br />
Through differential hybridization and sequencing of approximately 300 clones that appeared to be<br />
relevant clones, large differences in gene expression differentially expressed (Diehl et al 2002). One study<br />
were identified between the trophozoite and with T. cruzi reported changes in gene expression that<br />
gametocyte stages. A microarray of known P. occur as T. cruzi trypomastigotes convert into<br />
falciparum cDNAs was constructed using expressed amastigotes and identified 38 genes upregulated in<br />
sequence tags harvested at five different stages of amastigotes (Minning et al 2003).<br />
erythrocyte development and revealed common<br />
patterns of expression of groups of genes involved in In the other kinetoplastid genus, Leishmania, several<br />
carbohydrate metabolism, adhesion/invasion and studies in two species have used microarray analysis.<br />
translation machinery (Ben Mamoun et al 2001). A In L. major, a randomly sheared genomic microarray<br />
was used to study the changes in gene expression
14 DNA Microarray in parasitic diseases<br />
JPD : Vol. 28 (1), 2004<br />
among log-phase promastigotes, metacyclic<br />
promastigotes and lesion amastigotes (Beverley et al<br />
2002). <strong>The</strong> microarray results showed upregulation in<br />
log phase of genes encoding ß-tubulin, histones and<br />
ribosomal proteins, while in metacyclics this included<br />
HASP/gene B, SHERP/geneD, META1 and HSP70.<br />
Similar results were found in comparisons of logphase<br />
promastigotes versus amastigotes. <strong>The</strong> same<br />
microarray was used to analyze the global changes in<br />
gene expression as procyclic promastigotes<br />
differentiated in vitro into metacyclic promastigotes<br />
(Saxena et al 2003). Using a L. major cDNA array,<br />
changes in transcript abundance <strong>for</strong> different<br />
developmental stages across L. donovani and L. major<br />
were compared (Almeida et al 2002). This analysis<br />
identified 13.5% unique genes that were significantly<br />
upregulated in amastigotes. Taking the microarray<br />
results, amastigote-expressed genes were. used in<br />
high-throughput DNA-vaccine screens to identify<br />
potential vaccine candidates. Gene expression in L.<br />
donovani was compared between cultured<br />
promastigotes and axenic amastigotes on a genomic<br />
microarray containing 2304 clones (Duncan, 2004).<br />
<strong>The</strong> results identified many clones from apparently<br />
"new" genes. <strong>The</strong> major contribution of these studies<br />
was in validation of the array and new gene discovery,<br />
but no pattern of coordinate expression of functionally<br />
regulated or pathway-specific genes was described<br />
(Duncan, 2004).<br />
MICROARRAY PROGRESS WITH<br />
TREMATODE AND NEMATODE PARASITES<br />
<strong>The</strong> first generation S. mansoni cDNA microarray<br />
consisted of 576 expressed sequence tags selected<br />
from three different cDNA libraries and originating<br />
from two different parasite developmental stages<br />
(Hoffmann et al 2002). <strong>The</strong> microarray hybridizations<br />
identified 12 novel female-associated and 4 new male-<br />
associated gene transcripts in the mature adult<br />
Schistosome.<br />
cDNA microarray was used to evaluate a discrete<br />
biological event related to the life cycle of Ascaris<br />
suum, a nematode parasite of swine (Morimoto et al<br />
th<br />
2003). Gene expression patterns of A. suum 4 -stage<br />
larvae from the jejunum of infected pigs at 21 days<br />
after inoculation showed that sequences from muscle<br />
actin and myosin, ribosomal protein L11,<br />
glyceraldehyde-3-phosphate dehydrogenase and the<br />
flavoprotein subunit of succinate dehydrogenase were<br />
highly expressed in this larval stage.<br />
Gender-specific gene expression was studied in<br />
Trichostrongylus vitrinus, which is an important<br />
nematode parasite of small ruminants in temperate<br />
climatic zones (Nisbet and Gasser, 2004). Microarray<br />
analysis demonstrated gender-specific expression <strong>for</strong><br />
518 of the 716 T. vitrinus expressed sequence tags.<br />
Genes involved in gametogenesis, embryogenesis,<br />
reproduction and different cellular processes were<br />
expressed in a gender-specific manner.<br />
CONCLUSION<br />
Expression profiling by microarray analysis has been<br />
reported from a small number of taxa, and no doubt the<br />
popularity of this technique will increase in<br />
parasitology as resources become more widely<br />
available (Ellis et al 2003). Complete genomic<br />
sequences of different parasites and hosts will offer<br />
sophisticated new strategies <strong>for</strong> studying host-<br />
pathogen 'interactions. Apart from the construction of<br />
DNA micrqarrays <strong>for</strong> parasite genome, host profiling<br />
might also identify gene expression signatures unique<br />
<strong>for</strong> each parasite. Despite the success of DNA<br />
microarrays in gene expression profiling, it is the<br />
activity of encoded proteins that directly manifest<br />
gene function. As protein function is a direct<br />
consequence of the protein product of the gene and as<br />
mRNA levels do not always correlate well with<br />
protein, it is desirable to analyse the entire protein<br />
complement of a cell etc. on a similar scale to mRNA<br />
(Cutler, 2003). In terms of applicability and ease of use<br />
in a microarray <strong>for</strong>mat DNA has several advantages<br />
over protein. Still, analysis of protein function and<br />
protein interactions at the genomic scale is becoming a<br />
reality, albeit at the moment restricted to lower<br />
eukaryotes. In the next few years, it is likely that the<br />
use of protein arrays <strong>for</strong> other organisms, especially<br />
parasites will be available with continued innovation.<br />
Although each technique brings with it both<br />
advantages and limitations, taking the technology of<br />
parasite DNA microarrays, host arrays and protein<br />
arrays into an integrated approach will provide a novel<br />
tool <strong>for</strong> diagnosis, prognosis, and clinical management<br />
of parasitic diseases.<br />
ACKNOWLEDGEMENTS<br />
Mr. A. Debnath is grateful to UNESCO-American<br />
<strong>Society</strong> <strong>for</strong> Microbiology <strong>for</strong> selecting Short-term<br />
International fellow and providing the travel grant to
JPD : Vol. 28 (1), 2004<br />
DNA Microarray in parasitic diseases<br />
15<br />
work at Sandler Center <strong>for</strong> Basic Research in Parasitic Cummings CA and Relman DA. 2000. Using DNA microarrays to<br />
Diseases, University of Cali<strong>for</strong>nia, San Francisco, CA study host-microbe interactions. Emerging Infectious<br />
Diseases. 6 : 513-525.<br />
94143, USA and <strong>The</strong> Sandler Family Supporting<br />
Foundation <strong>for</strong> supporting the E. histolytica Cutler P. 2003. Protein arrays: the current state-of-the-art.<br />
microarray work. Mr A. Debnath and Mr. A. Sen are<br />
Proteomics. 3 : 3-18.<br />
also grateful to ICMR and DBT, Govt. of India Debnath A, Das P, Sajid M and McKerrow JH. 2004. Identification<br />
respectively, <strong>for</strong> financial assistance provided to them ofgenomic responses to collagen binding by trophozoites of<br />
Entamoeba histolytica. Journal of Infectious Diseases.190: 00<br />
during their stay at-NICED, Kolkata, India. Adam<br />
(in press).<br />
Carrol of the UCSF Genomics and Proteomics Core<br />
provided invaluable aid and discussion Degrave WM, Melville S, Ivens A and Aslett M. 2001. Parasite<br />
genome initiatives. International Journal <strong>for</strong> <strong>Parasitology</strong>. 31 :<br />
(http://derisilab.ucsf.edu/core/). 532-536.<br />
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Journal of Parasitic Diseases<br />
Vol. 28 (1) June, 2004, pp. 17-22<br />
Epidemiological studies on bovine microfilariasis in coastal<br />
districts of Andhra Pradesh<br />
V. PAVAN KUMAR, B. SREEDEVI*, T. VENKATA REDDY AND K. NALINI KUMARI<br />
Department of Veterinary Epidemiology and Preventive Medicine,<br />
College of Veterinary Science, Tirupati - 517 502. Andhra Pradesh<br />
In order to record the prevalence of bovine microfilariasis. 2399 blood samples from bovines (2168 buffaloes<br />
and 231 cattle) in the coastal districts (Tanuku, Mandapeta and Gudiwada) and Tirupati were screened<br />
during the period of November 2001 to June 2002-<strong>The</strong> overall prevalence recorded in bovines was 4.29 per<br />
cent (103 / 2399). <strong>The</strong> prevalence observed in buffaloes and cattle was 4.43 per cent (96/2168) and 3.03 per<br />
cent (7/231) respectively. Highest prevalence was observed in coastal areas, that is Tanuku (5.61%), followed<br />
by Mandapeta (3.30%) and Gudiwada (3.22%). whereas in Tirupati, the prevalence observed was only 1.92<br />
per cent. Animals of above nine years age group were found to be more prone <strong>for</strong> the disease (9.54%),<br />
followed by the age groups of six to nine years (4.83%) and three to six years (2.16%), while a lowest<br />
prevalence (0.88%) was recorded in below three years age group. <strong>The</strong> prevalence noticed in female was 4.32<br />
per cent (103/2382) whereas no positive case could be detected among the 17 male animals screened. With<br />
regards to breed wise analysis, highest prevalence was observed in graded Murrah (5.21%) than nondescriptive<br />
(2.95%) buffaloes. Testing of white cattle <strong>for</strong> microfilariasis revealed an prevalence of 3.57<br />
(7/196) and nil (0/35) in cross bred and indigenous cattle respectively A higher rate of prevalence of the<br />
disease was noticed in lactating animals (5.46%) followed by non-lactating animals (2.67%) and heifers<br />
(1.53%). No case was recorded among the calves and adult males screened. Highest prevalence of the disease<br />
was noticed during monsoon (3.77%) followed by summer (3.14%) and the least prevalence during winter<br />
(2.11 %).<br />
Key words : Andhra Pradesh, Bovines, Epidemiology, Microfilariasis<br />
I<br />
nfectious diseases have been identified as one of the<br />
major factors disrupting the development of dairy<br />
industry in India. Among them parasitic diseases even<br />
though are not life threatening, jeoparadise the<br />
economy of farmers. Among various parasitic diseases<br />
of livestock, filariasis is an important entity with a<br />
worldwide distribution. Filariasis caused by<br />
nematodes belonging to the filariodea consists of<br />
important genera, which include Setaria, Onchocerca,<br />
Stephana filaria, Parafilaria and Elaeophora. Adult<br />
worms are inhabitants of the peritoneal cavity of the<br />
animals without causing any adverse effects. But the<br />
infective larval stage (L3 <strong>for</strong>m), of these adult worms<br />
known as microfilariae cause a clinical condition<br />
'microfilariasis'. It inflicts economic loss indirectly<br />
due to gradual deterioration in general body condition,<br />
marked decline in milk yield, prolonged convalescent<br />
*Corresponding Author<br />
period and treatment costs. <strong>The</strong> present investigation<br />
was carried out to study the epidemiology of bovine<br />
micro filariasis including its prevalence with respect to<br />
species, age, breed, sex, physiological status and<br />
season.<br />
MATERIALS AND METHODS<br />
<strong>The</strong> present study was carried out in West Godavari,<br />
East Godavari, Krishna districts and Tirupati (Chittoor<br />
district) to assess the prevalence of bovine<br />
microfilariasis. <strong>The</strong> blood samples from all age<br />
groups, both sexes and animals in different<br />
physiological status were collected from buffaloes and<br />
cattle. <strong>The</strong> samples were screened <strong>for</strong> microfilariasis.<br />
Blood samples and smears were subjected to different<br />
diagnostic methods namely wet blood film<br />
examination, thick blood smear examination<br />
(Chatterjee, 1981) and modified Knot's method(<br />
Vihan. 1991). Among these three tests modified knot's
18 Epidemiological studies on boine microfilariasis<br />
JPD : Vol. 28 (1), 2004<br />
method and thick blood smear method were found to<br />
be more sensitive. Hence, in this sero epidemiological<br />
survey modified Knot's method was followed.<br />
Staining : For identification of the species of<br />
microfilariae and study of various morphological<br />
structures Giemsa and JSB stainings were followed. <strong>The</strong><br />
morphological characterisation of the microfilaria was<br />
done according to the method described by Sloss et al.<br />
(1994). <strong>The</strong> micro filaria identified in the present study<br />
were presumed to be the larval <strong>for</strong>ms of Seteria cervi.<br />
Study of periodicity : <strong>The</strong> periodicity pattern of the<br />
microfilaria was studied at every three hrs interval <strong>for</strong><br />
a duration of 24 hrs. <strong>The</strong> peak concentration was<br />
observed at 9 pm. <strong>The</strong> bovine population presented in<br />
the Veterinary institutions in these areas during the<br />
period of November 2001 to June 2002 were screened<br />
<strong>for</strong> microfilariasis by different methods of diagnosis.<br />
<strong>The</strong> details regarding the study population are given in<br />
a tabular <strong>for</strong>m (Table I).<br />
Epidemiological data has been collected with respect<br />
to host, agent and environmental factors. Data was<br />
collected in a prescribed pro<strong>for</strong>ma with the following<br />
parameters like place, date, owner's name, case<br />
number, species, age, sex, season, clinical signs, milk<br />
production, agro-climatic conditions (temperature and<br />
relative humidity), geographical in<strong>for</strong>mation with<br />
respect to surroundings and vector population. <strong>The</strong><br />
statistical analysis of the data was carried out by<br />
using the standard methods of Snedecor and<br />
Cochran. (1994).<br />
RESULTS AND DISCUSSION<br />
Area wise prevalence : <strong>The</strong> area wise prevalence of<br />
the disease observed was 5.61 per cent (63/1123), 3.30<br />
Prevalence : <strong>The</strong> prevalence of bovine microfilariasis<br />
was studied in Tanuku (West Godavari), Mandapeta<br />
(East Godavari), Gudiwada (Krishna) and Tirupati<br />
(Chittoor). In the present study totally 2399 bovines<br />
were screened. In this 2168 buffaloes and 231 cattle<br />
were screened <strong>for</strong> microfilariasis between November-<br />
2001 to June-2002. <strong>The</strong> overall prevalence recorded<br />
was 4.29 per cent (103 / 2399). Similar finding was<br />
reported by Sastry et al. (1985) who recorded an<br />
overall prevalence of 5.25 % in East Godavari district,<br />
Andhra Pradesh.<br />
per cent (10/303), 3.22 per cent (28/869) and 1.92 per<br />
cent (2/104) in Tanuku, Mandapeta, Gudiwada and<br />
Tirupati respectively (Table I). Higher prevalence was<br />
recorded in coastal areas than the dry areas. This may<br />
be due to swampy ness of these areas with a yearlong<br />
high humid climate. Urquhart et al. (1996) reported<br />
that the prevalence of the disease will be more in<br />
warmer areas where there will be long seasonal<br />
activity of the vector population.<br />
Species wise prevalence : Species wise prevalence<br />
noticed was 4.43 per cent (96/2168) and 3.03 per cent<br />
(7/231) in buffaloes and cattle respectively. This<br />
difference in prevalence rates was statistically not<br />
significant (Table 1). Similar prevalence rate was<br />
observed by Sastry el al.. (1985), Satish. (1996) and<br />
Venu et al. (2000).<br />
Age wise prevalence : Analysis of data in the present<br />
study revealed a highest prevalence in the animals of<br />
above nine years (9.54%) of age followed by groups of<br />
six to nine years (4.83%) and then three to six years<br />
(2.16%) age (Table II). Least prevalence was<br />
observed in animals below three years (0.88%) of<br />
age group. This finding is in accordance with the<br />
reports of Kumar et al. (1993) and Sharma and<br />
Kumar (1994) who recorded highest prevalence in<br />
animals aged above 12 years.<br />
Statistically there was no significant difference in the<br />
prevalence between below three years and three to six<br />
years of age groups. However, there was significant<br />
difference (P < 0.01) in the prevalence between below<br />
three years and other age groups (six to nine years and<br />
above nine years). <strong>The</strong>re was significant difference in<br />
the prevalence between three to six and six to nine<br />
years of age at one per cent level and six to nine and<br />
above nine years of age groups at five per cent level.<br />
Breed wise prevalence : Out of 1421 graded Murrah<br />
buffaloes screened <strong>for</strong> microfilariasis. 74 were found<br />
to be positive with an prevalence of 5.21 per cent<br />
(74/1421). In non-descriptive buffaloes, 22 out of 747<br />
(Table III) were positive <strong>for</strong> microfilariasis with an<br />
prevalence of 2.95 per cent (22/747). Normal deviate<br />
test <strong>for</strong> proportions of prevalence between the breeds<br />
revealed Z-value of 2.65 indicating higher prevalence<br />
in graded Murrah buffaloes, which was statistically<br />
significant (P < 0.01). This finding corroborates the
JPD : Vol. 28 (1), 2004<br />
Epidemiological studies on boine microfilariasis<br />
19<br />
Table I : Areawise and specieswise prevalence of bovine microfilariasis<br />
Number of bovines screened <strong>for</strong> Number of bovines screened <strong>for</strong> Percentage prevalence<br />
Area microfilariasis microfilariasis Z value<br />
Cattle Buffaloes Total Cattle Buffaloes Total Cattle Buffaloes Total<br />
Tanuku (West Godavari) 102 1021 1123 5 58 63 4.90 5.68 5.61<br />
Mandapeta (East Godavari) 18 285 303 0 10 10 0.00 3.51 3.30<br />
Gudiwada (Krishna) 42 827 869 - 28 28 0.00 3.39 3.22<br />
Tirupati (Chittoor) 69 35 104 2 - 2 2.90 - 1.92<br />
NS NS NS<br />
Total 231 2168 2399 7 96 103 3.03 4.43 4.29 1.15<br />
NS : Not significant<br />
Table II : Agewise prevalence of bovine microfilariasis<br />
Particulars < 3 years 3-6 years 6-9 years > 9 years Z - value<br />
Number of bovines screened <strong>for</strong> microfilariasis 113 741 1304 241 Z<br />
1.2<br />
= 1.24<br />
NS<br />
Z<br />
1.3<br />
= 3.71**<br />
Number of positives <strong>for</strong> microfilariasis 1 16 63 23 Z 1.4 = 4.15**<br />
Z 2.3 = 3.35**<br />
Percentage incidence 0.88 2.16 4.83 9.54 Z<br />
2.4<br />
= 3.76**<br />
Z<br />
3.4<br />
= 2.38*<br />
NS : Not significant ** Significant at P < 0.01 * Significant at P < 0.05
20 Epidemiological studies on boine microfilariasis<br />
JPD : Vol. 28 (1), 2004<br />
Table III : Breedwise prevalence of bovine microfilariasis<br />
Buffaloes Cattle<br />
Particulars Graded MurrahNon-Descriptive Total Cross bred Indigenous Total Z-value<br />
(1) (2) (3) (4)<br />
Number of animals screened <strong>for</strong> microfilariasis 1421 747 2163 196 35 231<br />
Number of positives <strong>for</strong> microfilariasis 74 22 96 7 0 7 Z<br />
1.2<br />
= 2.65**<br />
Percentage incidence 5.21 2.95 4.43 3.57 - 3.03<br />
** Significant at P < 0.01<br />
Table IV : Sexwise prevalence of bovine microfilariasis<br />
Particulars Females Males Total<br />
Number of bovines screened <strong>for</strong> microfilariasis 2382 17 2399<br />
Number of positives <strong>for</strong> microfilariasis 103 - 103<br />
Percentage incidence 4.32 - 4.29
JPD : Vol. 28 (1), 2004<br />
Epidemiological studies on boine microfilariasis<br />
21<br />
Table V : Prevalence of bovine microfilariasis with respect to physiological status<br />
Females Males<br />
Particulars Calves Heifers Lactating Non-lactating Calves Adults Z-value<br />
animals animals<br />
(1) (2) (3) (4) (5) (6)<br />
Number of animals screened <strong>for</strong> microfilariasis 39 131 1501 711 6 11 Z2.3 = 3.22**<br />
Number of positives <strong>for</strong> microfilariasis - 2 82 19 - - Z2.4 = 0.93NS<br />
Percentage incidence - 1.53 5.46 2.67 - - Z3.4 = 3.31**<br />
NS : Not significant ** Significant at P < 0.01<br />
Table VI : Seasonwise prevalence of bovine microfilariasis (April 2001 to March 2002)<br />
Particulars No. of bovines screened No. of positives Percentage incidence Z-value<br />
(1) (2) (3)<br />
Summer (March - June) 1719 54 3.14 Z1.2 = 1.10NS<br />
Monsoon (<strong>July</strong> - October) 2360 89 3.77 Z1.3 = 1.92NS<br />
Winter (November - February) 1850 39 2.11 Z2.3 = 3.23**<br />
NS : Not significant ** Significant at P < 0.01
22 Epidemiological studies on boine microfilariasis<br />
JPD : Vol. 28 (1), 2004<br />
observation made by Satish. (1996) and Kumar et al. rainy season. In contrast. Mukherjee (1965) recorded<br />
(1993) who reported higher prevalence in graded higher prevalence during summer and lowest in rainy<br />
Murrah buffaloes, than non-descriptive buffaloes. seasons.<br />
Out of 196 crossbred cattle screened <strong>for</strong><br />
microfilariasis. seven were positive with an<br />
REFERENCES<br />
Chatterjee KD. 1981. <strong>Parasitology</strong> (Protozoology and<br />
prevalence of 3.57 per cent (7/196). In case of 35 Helminthology) in relation to Clinical Medicine 12th Edn.<br />
indigenous cattle screened, none was found to be Chatterjee Medical Publishers. Calcutta.<br />
positive <strong>for</strong> microfilariasis.<br />
Kumar M, Joshi HC and Garg SK. 1993. Prevalence of Setciriti<br />
Sex wise prevalence : Among 2382 female animals<br />
cen-i in buffaloes of Kumaon region. <strong>Indian</strong> Journal of<br />
Veterinary Research. 2 : 1-6.<br />
screened <strong>for</strong> bovine microfilariasis 103 were found to<br />
Mukherjee RP. 1965. Percentage of microfilarial infections and the<br />
be positive with an prevalence of 4.32 per cent period of their maximum concentration in the blood of<br />
(103/2382). whereas out of 17 male animals screened, buffaloes. <strong>Indian</strong> Veterinary Journal. 42 : 131-133.<br />
none were positive (Table IV). However, no Sastry GJS, Rao VP, Narsimham MVVL and Rao PK. 1985.<br />
conclusion could be drawn on sex wise prevalence due Animal filariasis in East Godavari district, Andhra Pradesh.<br />
to small sample size.<br />
Livestock Adviser. 10: 56-60.<br />
Prevalence with respect to physiological status :<br />
<strong>The</strong> observations pertaining to the prevalence of<br />
microfilariasis in this study showed highest<br />
prevalence in lactating animals (5.46%) followed by<br />
non-lactating animals (2.67%) and heifers (1.53%).<br />
No case was recorded in calves and adult male<br />
buffaloes (Table V). <strong>The</strong> normal deviate test <strong>for</strong><br />
proportions of prevalence between heifers and<br />
lactating animals: between lactating and non-lactating<br />
animals showed a significant difference (P < 0.01)<br />
indicating highest prevalence in lactating animals.<br />
However, there was no significant difference in the<br />
prevalence between heifers and non-lactating animals.<br />
Satish Udhav, Rao Digrasakar. 1996. Clinico therapeutic studies<br />
on Microfilariasis in Buffaloes (Biihualus huhulis). Ph.D.<br />
<strong>The</strong>sis. Acharya NG Ranga Agricutlural University,<br />
Rajendranagar. Hyderabad.<br />
Sharma MC. 1985. Patho-biochemical and therapeutic studies on<br />
filariasis in cattle, buffaloes and dogs Ph.D. <strong>The</strong>sis. G B Pant<br />
University of Agriculture and Technology. Pantnagar.<br />
Sharma SP and Kumar M. 1994. Studies on the prevalence of<br />
clinical Setaria infection in buffaloes and horses. <strong>Indian</strong><br />
Veterinary Journal. 71: 1243-1245.<br />
Sloss MW, Kemp RL and Zajac AM. 1994. Veterinary Clinical<br />
<strong>Parasitology</strong> 6th Edn. Iowa State University Press. Ames. India,<br />
Snedecor GW and Cochran WG. 1994. Stalisficul methods. 8th<br />
Edn. Ox<strong>for</strong>d and IBH Pub. Co. Pvt.Ltd. Calcutta.<br />
Urquhart GM. Armour J. Duncan JL. Dunn AM and Jennings FW.<br />
Season wise prevalence : <strong>The</strong> yearlong data (April 1996. Veterinary <strong>Parasitology</strong> lind Edn. Blackwell Science Ltd.<br />
2001 to March 2002) collected from the veterinary<br />
Venu R. Radhakrishna Murthy P and Sreedevi C. 2000. Prevalence<br />
institutions (Tanuku, Mandapeta and Gudiwada) was<br />
ofmicrofilariasis in graded Murrah buffaloes in West Godavari<br />
subjected to analysis <strong>for</strong> studying the prevalence of district of Andhra Pradesh. <strong>Indian</strong> Veterinary Journal. 77: 272-<br />
microfilariasis with respect to season. <strong>The</strong> highest 273.<br />
prevalence (3.77%) was recorded during monsoon Vihan VS. 1991. Modern Veterinary Laboratory Techniques in<br />
(<strong>July</strong> - October). <strong>The</strong> prevalence in summer (March -<br />
June) was next to monsoon showing percentage<br />
prevalence of 3.14 (Table VI). Least prevalence<br />
(2.118%) was observed during winter (November -<br />
February). Statistically however, there was no<br />
significant difference in prevalence during summer<br />
and other seasons (i.e. monsoon and winter). whereas<br />
there was significant difference (P < 0.01) in the<br />
prevalence during monsoon and Winter. Sharma<br />
(1985) and Satish. (1996) reported peak prevalence in<br />
Clinical Diagnosis 1st Edn. CBS Publishers and Distributors.<br />
Delhi.
Journal of Parasitic Diseases<br />
Vol. 28 (1) June 2004, pp. 23-28<br />
Seasonal occurrence of helminth parasites in Schizothorax<br />
in Dal Lake Kashmir<br />
A. R. KHAN*, M. Z. CHISHTI, FAYAZ AHMAD, MAJIDAH RASHID & SHAFQAT BAKSHI<br />
Post Graduate Department of Zoology, University of Kashmir, Srinagar, India: 190 006<br />
Helminth parasitization is one of the major factors responsible <strong>for</strong> the decline of fish fauna of the valley, which<br />
provides protein to almost 60% of the people of valley in addition to providing sport and employment to Parge<br />
population particularly in rural areas. <strong>The</strong> present paper is an attempt to highlight the occurrence of different<br />
helminth groups in different seasons in order to find out a relationship between the helminth occurrence and<br />
season supported by data spreading over more than 3 years from 1991-93 and again from 1997-1998 In all four<br />
helminth groups including Trematoda, Cestoda, Nematoda and Acanthocephala infecting the native fish-<br />
Schizothorax, were studied represented in the present studies by 9 genera including Diplozoon, Clinostomum<br />
and Allocreadium from Trematoda; Adenoscolex from Cestoda; Pomphorhynchus, Ncoechinorhyncus and<br />
Tenuiproboscis from Acanthocephala and Rhabdochona from Nematoda group. About 610 Schizothorax<br />
specimens from Dal lake were studied through regular periodical collections. <strong>The</strong> hosts were scanned <strong>for</strong><br />
helminth presence and the data analysed <strong>for</strong> seasonal occurrence of the helminths. <strong>The</strong> tables (1-4) clearly<br />
depict the seasonal occurrence of helminth parasites indicating that season plays a vital role in the fish<br />
parasitization.<br />
Key Words: Fish; Helminths; Seasonality.<br />
INTRODUCTION<br />
Total number of hosts infected<br />
Prevalence = --------------------------------- xl00<br />
he population of Schizothorax is declining in the<br />
Twaters of Kashmir. <strong>The</strong> reasons may be varied, but<br />
Total number of hosts examined<br />
helminth infestation is an important factor responsible<br />
<strong>for</strong> this decline. In order to conserve this native fish,<br />
Total number of Parasites<br />
studies from all corners is important and the present<br />
Mean intensity = ------------------------------------------<br />
study deals with .the occurrence of helminth parasites in<br />
Total number of hosts infected<br />
different seasons in an urban lake-<strong>The</strong> Dal lake of<br />
Total number of parasites<br />
Kashmir. Relative density = -----------------------------------------<br />
or Abundance Total number of hosts examined<br />
MATERIALS & METHODS :<br />
Five hosts belonging to the native genus Schizothorax<br />
were collected alive fresh <strong>for</strong>tnightly from fishermen in<br />
Dal Lake & analysed in the laboratory through the<br />
routine Parasitological techniques. <strong>The</strong> following<br />
ecological terms as given by Morgoll's et al (1982) are<br />
used in the present study.<br />
* Corresponding Author<br />
OBSERVATION<br />
Schizothorax was sampled throughout the three years in<br />
Dal lake and a total of 600 specimen were investigated<br />
<strong>for</strong> helminlhological survey. <strong>The</strong> occurrence of<br />
different helminth parasites in the four seasons was<br />
studied. <strong>The</strong> helminthological patterns of this host<br />
observed parasite-wise in different seasons were as<br />
follows :
24 Seasonal occurrence of helminth parasities in fish<br />
JPD : Vol. 28 (1), 2004<br />
Trematoda - Diplozoon<br />
44 Acanthocephalan parasites belonging to the genus<br />
Pomphorhynchus. <strong>The</strong> infection again showed a<br />
Out of the three trematode genera viz. Diplozoon,<br />
seasonal occurrence, as the fishes were found to be<br />
Clinostomum and Allocreadium recorded during the<br />
infected either during spring or early summer, while <strong>for</strong><br />
present investigation in different fish hosts, the rest of the survey period no infection of the parasite was<br />
infection of the only ectoparasitic genus Diplozoon was<br />
recorded as shown in table III.<br />
recorded in this host from Dal lake. Whereas<br />
Clinostomum and Allocreadium were absent. <strong>The</strong> fishes <strong>The</strong> prevalence of infection of Pomphorhynchus was<br />
were invariably found to be infected with the Diplozoon highest i.e. 5. 74 during spring and the minimum of 1. 14<br />
parasite i.e. Diplozoon kashmirensis, although M. 1. during summer season. Minimum M. 1. of 1.5 during<br />
and R. D. showed variations as shown in the table I. summer and maximum M. 1 (3. 41) during spring.<br />
Whereas the R. D. of 0.196 was observed during spring<br />
<strong>The</strong> prevalence of infection of D, Kashmirensis in<br />
and 0. 017 during summer.<br />
Schizotliornx was the highest (19.61) during spring,<br />
while as the minimum value of prevalence was recorded Nematoda - Rhabdochona<br />
in the autumn season (i.e. 9.70) . <strong>The</strong> highest M. I. of<br />
3.46 was recorded during autumn and the minimum of<br />
<strong>The</strong> infection of Nemaloda in Schizothorax. from Dal<br />
1.92 during summer. Maximum R.D. of 0.55 was<br />
Lake during tlie present 3 years of investigation was<br />
observed in spring season and minimum of 0.29 in<br />
restricted to the spring season. While <strong>for</strong> rest of the<br />
summer season .<br />
season of the year the infection was altogether absent.<br />
Cestoda - Adenoscolex oreini<br />
<strong>The</strong> prevalence of Rhabdochona was the minimum out<br />
of the four groups of parasites recorded so far and<br />
Out of the total of 610 fishes from Dal Lake only five restricted to a short period of late spring, the prevalence<br />
fishes were found to be infected with 9 parasites being 1.91 in spring, M. I. being 2 and R. D. 0. 03 as<br />
belonging to the cestode genus Adenoscolex . Cestode shown in table IV.<br />
infection showed a marked seasonal occurrence as the<br />
infection was observed only during spring and summer<br />
DISCUSSION<br />
seasons as is clear from the Table II.<br />
Studies on seasonal dynamics of fish parasites with respect<br />
<strong>The</strong> prevalence of infection of Adenoscolex being 1.43<br />
to the fluctuations of the parasitic fauna are meager.<br />
during spring; and 1.14 during summer. M. 1. of<br />
Williams (1963, 1979), Chubbs and Thomas (1964).<br />
infection was 2.5 during summer season and 1.33<br />
Kennedy (1969), Tedia (1970), Hare and Burt (1974-75),<br />
during spring R. D of 0.02 during summer and 0. 019<br />
and Cling (1988), Carried out studies on the seasonal<br />
during spring.<br />
influence on the helminth parasites and concluded that,<br />
season plays a significant role in the prevalence and<br />
Acanthocephala - Pomphorhynchus<br />
intensity of helminth parasitization of fish.<br />
A total number of 14 Schizothorax out of 610 fishes<br />
from Dal Lake dissected were found to be infected with<br />
In the present study following results on four helminth<br />
groups were arrived at :<br />
Table I: Seasonal Changes of Infection pattern of D. kashmirensis<br />
Seasons No. of No. of %age No. of M. I. R.D.<br />
fishes fishes infected parasites<br />
exam. infected<br />
Summer 175 27 15.42 52.0 1.92 0.29<br />
Autumn 134 13 9.70 45 3.46 0.33<br />
Winter 92 14 15.21 41 2.92 0.44<br />
Spring 209 41 19.61 115 2.80 0.55
JPD : Vol. 28 (1), 2004<br />
Seasonal occurrence of helminth parasities in fish<br />
25<br />
Trematoda<br />
From this group the ectoparasitic monogenetic<br />
trematode Diplozoon was found to be infecting this fish<br />
host genus, although the occurrence and intensity<br />
fluctuated considerably. Evans (1978) while studying<br />
the trematodes, observed that the prevalence and<br />
intensity of infection increased during the spring and<br />
summer and by <strong>July</strong> and August the majority of<br />
parasites were also fully mature.<br />
A marked seasonal effect was noted, being most<br />
abundant during the summer on the gill parasites of<br />
Lcpomis, by Ilanck (1978), Meyers (1978) and Dronen<br />
(1978).<br />
Glenn (1980) observed that the prevalence of infection<br />
of Crepidostomum hiodontis (Trematode) increased in<br />
August and September to a relatively high constant<br />
winter rate of approximately 86%. This tremalode was a<br />
common parasite of both age groups of moon eye. For<br />
the older age C. hiodontis was found during each month,<br />
prevalence was lost in June and <strong>July</strong>, increased in<br />
August and was relatively constant during the autumn<br />
and winter. But Palurorhynchus hiodntis in infected fish<br />
varied from peaks in summer to lows in winter.<br />
Table II: Seasonal Changes of Infection pattern of Adenoscolex<br />
Seasons No. of No. of %age No. of M. I. R. D<br />
fishes fishes infected parasites<br />
exam. infected<br />
Summer 175 2 1.14 5 2.5 0.02<br />
Autumn 134 - - - - -<br />
Winter 92 - - - - -<br />
Spring 209 3 1.43 4 1.33 0.019<br />
Table III: Seasonal Changes of Infection pattern of Pomphorhynchus<br />
Seasons No. of No. of %age No. of M. I. R. D.<br />
fishes fishes infected parasites<br />
exam. infected<br />
Summer 175 2 1.14 3 1.5 0.017<br />
Autumn 134 - - - - -<br />
Winter 92 - - - - --<br />
Spring 209 12 5.74 41 3.41 0.196<br />
Table IV: Seasonal Changes of Infection pattern o f Rhabdochona<br />
Seasons No. of . No. of %age No. of M. I. R. D<br />
fishes fishes infected parasites<br />
exam.<br />
infected<br />
Summer 175 - - - - -<br />
Autumn 134 - - - - -<br />
Winter 92 - - - - -<br />
Spring 209 4 1,91 8 2 0.03
26 Seasonal occurrence of helminth parasities in fish<br />
JPD : Vol. 28 (1), 2004<br />
Muzzal (1980) while working on the trematode<br />
Triganodistomum attenuatum, recorded peak in spring<br />
season.<br />
Recruitment of Uvulifer ambloplitis by caged blue gill<br />
began in May and ended in September, with the<br />
maximum recruitment occurring in <strong>July</strong> as reported by<br />
Dennis and Gerald (1984),while working on population<br />
biology of trematodes.<br />
Dennis and Esch (1985) after working on the<br />
trematodes observed peak in <strong>July</strong> (early summer). <strong>The</strong>y<br />
also supported the significant role of season in the<br />
infection of helminth parasites.<br />
Rand and Burlt (1985) supported the role of season in<br />
the infection of parasites while working on the seasonal<br />
occurrence, recruitment of Allocreadium lobatum<br />
showing that the prevalence of infection increasing to<br />
high levels from November through May.<br />
According to James (1988) size of the host plays a great<br />
role in the infection of parasites as large hosts carried<br />
significantly more Eirocotyle spinicirrus individual<br />
than smaller ones and highest infection <strong>for</strong> larger<br />
individuals was observed in May, where as the smaller<br />
length classes were dominant from June through<br />
October 1986.<br />
definitive host-lhc dace Leuciscus leuciscus. Infection<br />
of fish is rcslriclcd to colder winter months and the<br />
parasite matures in late spring and winter be<strong>for</strong>e<br />
disappearance in summer. Kennedy and Walker (1969)<br />
have shown experimentally that at low temperature C.<br />
laticeps is better able to establish itself in the Dace and<br />
survives <strong>for</strong> longer periods than at high temperature.<br />
Thus supporting the results arrived during the present<br />
investigation.<br />
Fischer and Freeman (1969) reported the incidence of<br />
enteric adult Proteocephalus ambloplitis in small<br />
mouth bass (Micropterus dolomievi) was highest in<br />
spring declining in the summer and disappearing in late<br />
autumn.<br />
After studying the trcmalodes Gyrodactylus cole-<br />
manensis , G. salmonis by Cone and Cusack (1988), it<br />
was observed that the intensity of infection during<br />
winter to a spring peak followed by a decrease during<br />
the summer months.<br />
<strong>The</strong> prevalence and mean intensity of Glaridacris<br />
catostomi and Glaridacris larvrei in Oyster river hosts<br />
was highest in spring (1975-76) and winter (1976-77).<br />
<strong>The</strong> mean intensity of G. castostomic in Bellamy river<br />
hosts was highest in spring 1975 and 1976 as reported<br />
by Muzzall(1980).<br />
Infra population of Adenoscolcx Oreni increased in<br />
winter season as reported by Dhar and Peerzada (1992),<br />
while working on the seasonal variation in the<br />
occurrence and maturation of Adenoscolex Oren<br />
Fotedar 1958 infecting some Cyprinid fishes ofWular<br />
lake (Kashmir) Hence the present observations arc in<br />
con<strong>for</strong>mity with Kennedy (1969), Muzzall (1980) and<br />
Dhar and Peerzada (1992) and Fisher and Freeman<br />
(1969) indicating that season plays a great role in<br />
helminth infections.<br />
<strong>The</strong> distribution of four species of Dactylogyrus on the Acanthocephala<br />
bream's gill is fairly similar and peak was in spring and<br />
<strong>The</strong> Acanthocephala group represented by two genera<br />
summer as reported by Dzika and Szymanki (1989).<br />
viz., Pomphorhynchus & Neoechinorhynchus again<br />
Thus the present observations are in accordance with showed a marked seasonal occurrence, as the peak<br />
those of Evans (1978), Rand & Burt (1985), Muzzall infection in all the recorded observations was in spring<br />
(1980), Cones & Cusack (1988), James (1988), While it or early summer season.<br />
shows variation with the work of Hanek (1978), Meyers<br />
<strong>The</strong> effect of temperature and other factors upon the<br />
(1978), Dennis and Esch (1985).<br />
establishment and survival of Pomphorhynchus laevis<br />
Cestoda<br />
in gold fish was studied by Kennedy (1972). He further<br />
observed that although the density of infection had no<br />
<strong>The</strong> only ceslodc genus infecting the fish hosts under effect upon the establishment of the parasite but a 12°C<br />
discussion includes the Caryophyllidean genus rise on water temperature resulted in 30% reduction of<br />
Adenoscolex. A seasonal occurrence of this cestode was infection. But the establishment of fish Acanthocephala<br />
observed with two infection period spring and summer is affected by temperature to a lesser extent than that of<br />
in Dal Lake.<br />
some fish cestodes. <strong>The</strong> degree of infection of various<br />
hosts with Acanthocephala parksidei and the<br />
Kennedy (1969) reported that Caryophyllaeus laticeps<br />
development stages of these Acanthocephala showed<br />
exhibits a well defined seasonal incidence cycle in its
JPD : Vol. 28 (1), 2004<br />
Seasonal occurrence of helminth parasities in fish<br />
27<br />
considerable seasonal variations. Huzinga (1980) and Arnin (1988).<br />
Frequency of infection and maturation was highest<br />
during the spring as reported by Amin (1975), which<br />
clearly supports the present observations.<br />
Muzzall and Bullock (1978) have shown that the<br />
prevalence of Neoechinorhynchus saginalus was high<br />
(above 44% in all months sampled) and fluctuated<br />
irregularly throughout, the sampling period in 1975-76,<br />
the prevalence declined from high in June to a low in<br />
January and March and exhibited a tendency to increase<br />
again towards <strong>July</strong>. <strong>The</strong> Prevalence of Acanthocephala<br />
dirus in the Creek Chubs and Cystacanths in isopods<br />
increased through the autumn and winter, peaked in<br />
Feb. 1976 (Isopod) and April 1976 (Chubs) as reported<br />
by Camp and Huizinga (1980) al'lcr studying the<br />
seasonal population interaction of A . dirus.<br />
Nematoda<br />
Nematode infection again showed seasonal occurrence<br />
showing peak availability towards the winter and spring<br />
season.<br />
<strong>The</strong> data analysed by Stromberg and Crites (1975)<br />
shows seasonal cycle in population structure, site<br />
selection, intensity of infection maturation and<br />
reproduction. Infection of Camallanus oxycephalus<br />
occurs during <strong>July</strong> and August with a resulting peak in<br />
population density during late summer and autumn.<br />
<strong>The</strong>y further concluded that the infection was generally<br />
more frequent and higher intensity in longer fish.<br />
Abundance of Cystidicoloides tenuissima in the<br />
infected fish varied from peaks in summer to lows in<br />
Muzzall (1980) studying the impact of season on<br />
winter, as worked out by Glenn (1980). C. tenuissima,<br />
prevalence and intensity of helminths concluded that<br />
the most common and abundant species in trout did not<br />
the mean intensity of Pomphorhynchus bulbocolli was<br />
exhibit a pronounced seasonal pattern in prevalence;<br />
highest in spring and summer 1975, spring 1976, and<br />
mean intensity, however, was highest in <strong>July</strong> 1982, 83 in<br />
winter 1976-77 while as Neoechinorhynchus cristatus<br />
both trout species. <strong>The</strong> intensity of C. tenuissima<br />
did not exhibit a seasonal cycle in maturation or in<br />
increased as trout become older and than decreased in<br />
length distribution.<br />
brook and brown trout 3 & 4 years of age respectively.<br />
<strong>The</strong> prevalence and mean intensity of Spinitectus<br />
<strong>The</strong> Neoechinorhynchus prolixoidcs studied were gracilis were highest in <strong>July</strong> 1982, 83 & 84.<br />
mostly recruited during late summer early autumn,<br />
reached sexual maturity by spring and continued its<br />
simultaneous reproductive activity and growth through<br />
the summer into the following autumn (Amin, 1986).<br />
He also observed the recruitment of N. Cylindratus in<br />
late summer and autumn. He further noted that the<br />
seasonal periodicity of Neoechinorhychus prolixoides<br />
bore basic similarities to that of N. cylindratus except<br />
that the number of juveniles was more, particularly in<br />
the spring.<br />
Amin (1987) observed that the intensity of infection<br />
increased from low in the autumn to higher levels in<br />
warmer months and reached a maximum usually in the<br />
summer after studying the ecology and host relationship<br />
of Pomiphorhynchus bulbocolli. Amin (1988) reported<br />
Leptorhynchoides thecatus from Micropteruus<br />
salmoidcs. He observed that the egg presence, worm<br />
recruitment and maturation appear to occur throughout<br />
the year with maximum recruitment in the autumn,<br />
maturity and reproduction in spring and summer.<br />
After studying the seasonality of infection and<br />
histopathology of Cystidicoides ephemeridarum, the<br />
researcher Green Wood and Bakcr (1987) observed that<br />
the mean intensity followed a typical seasonal trend in<br />
salmonids. In both years it increased from May to reach<br />
a maximum in June and then decline considerably in<br />
<strong>July</strong> and August. <strong>The</strong>y also concluded that the season<br />
played a significant role in the intensity of infection.<br />
Thus the present observations show variations with<br />
Stromberg and Crites (1975), Glcnn (1980) and<br />
Greenwood and Baker (1987).<br />
CONCLUSION<br />
From the above account it is clear that season plays a<br />
vital role in helminth infestations although the effect is<br />
different on different helminth groups which may be<br />
related to their life cycle patterns e.g. recruitment,<br />
intermediate hosts etc. Inspitc of resemblances of the<br />
present observations with some previous researchers,<br />
there is a noticeable variation with others. <strong>The</strong> variation<br />
Thus the present observations are supported by Amin may be due to the different water body, host, feeding<br />
(1975, 1986 and 1987); Muzzall and Bullock (1978); habits of the host, diversity of the climatic conditions<br />
Muzzall (1980); while it goes against those of Camp and and availability of intermediate hosts. This warrants the
28 Seasonal occurrence of helminth parasities in fish<br />
JPD : Vol. 28 (1), 2004<br />
study of life cycle in these parasites and the detailed James, E. Joy, 1988 Monthly length class frequencies of Microcotyle<br />
study under experimental conditions in order to find out spinicirrus (Monogenea : Microcotylidae) from the freshwater<br />
drum, Aplodinotus grunniens, in west Virginia. Proc.<br />
the possible role of a particular factor in the recruitment Helminthol. Soc. Wash., 55 (2) : 246-251.<br />
and maturation of a particular parasite<br />
Kennedy, C.R., 1969 Seasonal incidence and development of the<br />
REFERENCES<br />
cestode, Caryophyllaeus laticeps (Pallas) in their river Avon. J.<br />
<strong>Parasitology</strong>, 59 : 783-794.<br />
Amin, O.M. 1975, Host and seasonal associated of Acanthocephalus<br />
parksidei Amin, 1974 (Acanthocephala: Echinorhynorhynchidae) in Kennedy, C.R., 1972 <strong>The</strong> effect of the temperature and other factors<br />
Wisconsin fishes. J. Parasitol., 61 (2): 318-329.<br />
up to the establishment and survival of Pomphorhynchus laevis<br />
(Acanthocephala) in gold Fish, Carassius suratus. J.<br />
Amin, O.M. 1986, Acanthocephala from lake fishes in Wisconsin <strong>Parasitology</strong>, 65 : 283-294.<br />
host and seasonal distribution of species of the genus<br />
Neoechinorhynchus Hamann, 118.<br />
Kennedy, C.R. and Walker, P.J., 1969 Evidence <strong>for</strong> an immune<br />
response by dace Leucisucus leuciscus, to infection by the<br />
Amin, O.M. 1987, Acanthocephala from lake fishes Wisconsin cestode Caryophyllaeus laticeps. J. <strong>Parasitology</strong>, 55 :579-582.<br />
(USA) : Ecology and host relationship of Pomphorhynchus<br />
bulbocoli (Pomphorhynehidae). J. Parasitol., 73 (2): 278-289. Meyer, T.R., 1978 Prevalence of fish parasitism in Raritan Bay, New<br />
Jersey. Proc. Helminthol. Soc. Wash.,45 (1): 120-128.<br />
Camp, J.W. Jr and Harry, W Hvizinga 1980 Seasonal population<br />
interaction of Acanthocephalus dirus (Van cleave, 1931) in the Margolis et al., 1982 <strong>The</strong> use of ecological terms in <strong>Parasitology</strong><br />
Creek Chubb, Semotilus astromaculatus and Isopod, Asellus (Report of an adhoc committee of the American <strong>Society</strong> of<br />
intermadius, J. Parasitol,. 66 (2) : 299-304.<br />
Parasitologists). J. Parasitol., 68 : 131-133.<br />
Ching, H.L. 1988 <strong>The</strong> distribution of Plerocercoides of Muzzall, P.M., 1980a Ecology and seasonal abundance of three<br />
Diphyllobothrium dendriticum (Nitzsch) in Sockeye Salmon Acanthocephalan species infecting white suckers in SE New<br />
(Onchorhynchus nerka) smotts from great central lake, British Hemsphire. J. Parasitol., 66 (1) 127-133.<br />
Columbia. Can. J. Zool., 66 : 850-852.<br />
Muzzall, P.M., 1980b Population biology and host-parasite<br />
Cone, D.K. and Cusack R, 1988 A study of Gyrodactylus colemanensis relationship of Triganodistomum attenuatum (Trematoda :<br />
Mizelle and Krilsky, 1967 and Gyrodactylus salmonmis Yin and Lissorchiidae) infecting the winter sucker, Catostomus<br />
Sproston, 1948 (Monogenca) Parasitizing captive salmonids in commersoni (Lacepede). J. Parasitol., 66 (2) 293-298.<br />
Wovw Scotia (Canada) Can. J. Zool, 66 (2): 409-415.<br />
Muzzall, P.M., 1980c Seasonal distribution and ecology of three<br />
Dhar, R.L. and Peerzada, M.Y. 1992 Seasonal variation in the occurrence Caryophyllac : Cestoda species infecting white suckers in SE<br />
and maturation of Adenoscolex Oceni Fotedar, 1958 (Cestoda : new Hemisphere. J Parasitol., 66 (3) : 542-550.<br />
Carophyllaeidae) infecting some Cyprinid fishes of Wular Lake Muzzall, P.M. and Wilburl Bullock, 1978 Seasonal abundance and<br />
(Kashmir). Current Trends in Fish and Fishery Biology and Aquatic host-parasite relationships Neoechinorhynchus saginatusi Van<br />
Ecology (Ed. Yousuf, Raina & Qadri) xv + 365.<br />
Cleave and Bangham, 1949 in the fall fish, Semotilus corporalis<br />
Dronen, Norman O., Jr., 1978 Host Parasite population dynamics of (Mitchill). J. Parasitol., 64 (5): 860-865.<br />
Haematolaechus (Digenea). Am. Midl. Nat., 99 (2) : 330-349. Rand, T.G. and M.D.B. Burt, 1985 Seasonal occurrence recruitment<br />
Dzika, E. and Stanislaw Szymanski, 1989 Co-occurrence and and maturation of Allocreadium lobatum Wallin, 1904 (Digenea :<br />
distribution of Monogenea of the genus Dactylogyrus on gills of Allocreadiidae) in the fall fish, Semotilus corporalis Mitchell, in a<br />
the bream, Abramis brama Acta, Parasitol, Pol., 34(1): 1-14.<br />
new brunswick, Canada Lake system. Can. J. Zool., 63: 612-616.<br />
Evans, N.A., 1978 <strong>The</strong> occurrence and life history of Asymphylodora Stromberg, P.C. and Crites, J.L., 1975 Population Biology of<br />
kubanicum (Platyhelminthes : Digenea : Monorchidae) in the Cammallanus oxycephalus Ward and Margath, 1916 (Nematoda<br />
Worcester - Birmingham canal, with special habits of definitive host, Cammallanidae) in white bass in western lake. J. Parasitol., 61 :<br />
Rutilus rutilus. J. Zool. Lond., 184 : 143-153.<br />
123-132.<br />
Glenn, C.L., 1980 Seasonal parasitic infections in moo eye, Tadla, S. and Fcrnando, C., II. 1970 Observations on seasonal<br />
Hiodon tergisus (Le Sueur), from the Assiniboine River. changes of the parasites of yellow perch from the Bay of Quinte,<br />
Cana. J. Zool., 58 : 252-257.<br />
Lake Ontario. Res. Board Can., 26 : 833-843.<br />
Greenwood, S.J. & Baker M.R., 1987 Cystidicoloedes Williams Dennis, D., 1979 Seasonal incidence of Isoglariducris<br />
wisconsincensis in its fish host (Cestoda : Caryophyllidac).<br />
ephermmeridarum (Linstow, 1872) (Nematoda) in speckled<br />
trout, Salvelinus frontinalis from southern Ontario (Canada).<br />
Can. J. Zool., 65 (II): 2589-2593.<br />
Hanek, G. and CH Fernando, 1978 Seasonal dynamics and spatial<br />
distribution of Urucleidus ferox uller 1934, a gill parasite of<br />
Lepomis gibbosus (L). Can. J. Zool., 56 : 1241-1243.<br />
IOWA Stale J. Res., 53 (4) : 305-310.<br />
William Dennis, D., 1979 Seasonal incidence of Glaridacris larvei<br />
and G. catastomi in Red cedar river, State J. Res., 53(4): 311-3 16.
Journal of Parasitic Diseases<br />
Vol. 28 (1) June 2004, pp. 29-36<br />
A Monoclonal Antibody to 120 kDA B.Malayi Antigen with<br />
Diagnostic Potential in Bancroftian Filariasis<br />
1 1<br />
BALAJI GANESH. B, PARAB P.B. , KATDARE. M , REDDY MVR AND HARINATH BC*<br />
Department of Biochemistry, JB Tropical Disease Research Centre, MGIMS, Sevagram, Wardha 442 102<br />
1<br />
& National Centre Cell sciences, Pune University Campus, Pune-411 007<br />
A monoclonal antibody (Bm Ab 120) of IgM isotype against B.malayi micro filarial soluble antigen [Bmmf<br />
(S)] that specifically recognized a 120 kDa antigen present in microfilarial soluble extracts, microfilarial<br />
excretory- secretory proteins and adult soluble proteins but not in soluble infective larval extracts was<br />
produced. Bm Ab 120 was employed in a sandwich ELISA to screen individuals in areas endemic <strong>for</strong><br />
bancroftian filariasis as well as those suffering from other parasitic infections from non-endemic area using<br />
two different sources of polyclonal antibodies viz, Filarial serum immunoglobulins (FSIgG) isolated from<br />
clinical patients and anti-Bmmf (S) antibodies raised in rabbits as captured antibodies. FSIgG- Bm Ab 120<br />
combination showed 84% mf carriers, 66% of patients with clinical symptoms of filariasis and 18% of<br />
endemic normals as positive <strong>for</strong> filarial antigens. When anti-Bmmf (S) antibodies were used as capture<br />
antibodies along with Bm Ab 120 antibodies as tracer probe, 88% of mf carriers were detected as also 54% of<br />
clinical cases and 12% endemic normals. A small percentage of patients with Tropical Pulmonary<br />
Eosinophilia were also picked up by both the assays. None of the sera of other parasitic infections such as<br />
ascaris, hookworm, leishmania and malaria showed levels above the cut-off suggesting high specificity of the<br />
monoclonal antibody. Thus, Bm Ab 120 was found to be useful in identifying significant numbers of<br />
asymptomatic microfilaraemics, along with patients suffering <strong>for</strong>m clinical and occult filariasis in<br />
bancroftian endemic areas.<br />
Keywords : Brugia malayi, Diagnosis, ELISA, Filariasis, Monoclonal antibodies, Wuchereria bancrofti,<br />
INTRODUCTION<br />
ymphatic filariasis is a cause of severe morbidity<br />
L<strong>for</strong> people living in filarial endemic areas with an<br />
estimated 120 million people suffering from this<br />
disease worldwide (WHO, 1994). It is caused by the<br />
blood- borne nematodes Wuchereria bancrofti, Brugia<br />
malayi and Brugia timori of which more than 90% of<br />
the cases are caused by W. bancrofti. <strong>The</strong> World Health<br />
Organization (WHO) has made it a goal to interrupt<br />
transmission and eliminate this disease globally by the<br />
year 2020 (WHO. 1993). <strong>The</strong> successful<br />
implementation of this ambitious programme would<br />
require the identification and treatment of individuals<br />
harbouring the infection. Besides microfilarial<br />
* Corresponding Author<br />
carriers, clinical and occult filarial patients (showing<br />
atypical manifestations) who are usually<br />
amicrofllaraemic but may harbour parasites in the<br />
internal organs or tissues would also be important<br />
targets <strong>for</strong> identification and treatment in the<br />
successful implementation of filaria control<br />
programmes.<br />
Specific monoclonal antibodies produced against<br />
antigens of mammalian stages of filarial parasites<br />
would act as suitable diagnostic agents <strong>for</strong> screening<br />
and identification of target population and could also<br />
have epidemiological implications. Lack of suitable<br />
animal models to maintain W. bancrofti infection has<br />
hampered availability of sufficient parasite material<br />
due to which heterologous antigens from other<br />
nematodes have been exploited to develop serological
30 Monoclonal antibody Bm Ab120 in diagnosis of filariasis JPD : Vol. 28 (1), 2004<br />
tests. Currently available diagnostic tools <strong>for</strong> antigen peritoneal lavage of infected jirds or at necropsy.<br />
detection include the ICT Filariasis and the Trop Bio Worms were first lyophilized, homogenized,<br />
ELISA that employ monoclonal antibodies directed sonicated and antigens extracted in PBS overnight.<br />
against Dirofilaria immitis and Onchocerca gibsoni <strong>The</strong> concentrated antigen was labeled as A (S) antigen.<br />
(both animal parasites) respectively and show high<br />
Immunization of mice : Six week old BALB/C mice<br />
sensitivity in detecting microfilarial carriers<br />
were immunized with 100 micro grams of Bmmf (S)<br />
(Chandrasena et al, 2002; Ramzy et al, 1999) but not<br />
antigen intra-peritoneally (i.p) on days 1, 8, 15 and 22.<br />
always in communities showing clinical<br />
<strong>The</strong> first immunization was with equal volume of<br />
manifestations of disease (Eigege et al, 2003; Nguyen nd rd<br />
Freunds complete adjuvant while the 2 and 3 doses<br />
et al, 1999). However, individuals showing prepatent<br />
th<br />
were with Freunds Incomplete adjuvant. <strong>The</strong> 4 and<br />
infection and mild or atypical manifestations of<br />
final dose was without any adjuvant. Animals that<br />
filariasis but absence of microfilaria in peripheral<br />
developed high antibody litres to Bmmf (S) as<br />
blood may escape detection by these assays. Diagnosis<br />
revealed by ELISA were used as donors of spleen cells<br />
of filarial etiology and morbidity control is equally<br />
<strong>for</strong> the production of hybridoma.<br />
important in filaria control programme. We there<strong>for</strong>e<br />
explored to develop a monoclonal antibody against Production of monoclonal antibodies (mAbs) : Cell<br />
microfilarial antigens of the closely related human fusion was per<strong>for</strong>med according to the method<br />
nematode, B. malayi, and evaluate the presence of described earlier (Galfre et a/, 1977), with minor<br />
these antigens in a spectrum of population living in an modifications. Briefly, spleen cells isolated from mice<br />
area endemic <strong>for</strong> bancroftian filariasis.<br />
immunized as above and SP2/0 myeloma cells<br />
MATERIAL AND METHODS<br />
(National Centre <strong>for</strong> Cell Science, Pune, India) were<br />
washed twice in serum-free Dulbecco's Modified<br />
Antigen preparations: Brugia malayi microfilariae Eagle Medium (DMEM, Gibco-BRL). <strong>The</strong> spleen<br />
were collected by the peritoneal lavage of B.malayi cells were mixed with myeloma cells at a ratio of 1:1<br />
infected jirds. <strong>The</strong> microfilariae were plated on sterile and were pelleted together at 400g <strong>for</strong> 5 min. Cell<br />
petri plates in order to allow the host peritoneal cells to fusion was promoted by the use of polyethylene glycol<br />
settle. <strong>The</strong>y were then maintained in vitro as described (PEG-1500, BDH chemicals, Ltd). <strong>The</strong> fused cell<br />
earlier (Alli et al., 2000) to prepare B.malayi<br />
6<br />
pellet was suspended to a concentration of 1 X 10<br />
microfilarial excretory-secretory antigens (Bm mf cells/ 0.5 ml in DMEM containing 10 % fetal calf<br />
ES). <strong>The</strong> parasites were then separated by serum and hypoxanthine, aminopterin and thymidine<br />
centrifugation, homogenized in ice in the presence of (HAT, Sigma Chemicals). This, suspension was<br />
cocktail protease inhibitors [0.1 mM ethylene diamine dispensed into 24- well flat-bottomed culture plates<br />
tetraacetic acid (EDTA) and Tosyl-L-lysine (0.5 ml/well) already containing mouse peritoneal<br />
chloromethyl ketone (TLCK)], sonicated and macrophages as a feeder layer and were incubated at<br />
extracted overnight at 4°C in 0.05M PBS and the 37°C in a humid atmosphere at 5% CO 2 in air. Culture<br />
homogenate was centrifuged at 12,000 rpm <strong>for</strong> 20 supernatants from wells displaying dense turbid cell<br />
minutes at 4°C. <strong>The</strong> supernatant was removed, growth were tested <strong>for</strong> the presence of antibody to<br />
concentrated by ultra membrane filtration and labeled Bmmf (S) by ELISA. Antibody-producing hybrids<br />
as Brugia malayi microfilarial soluble antigen were then cloned by a serial dilution technique on a<br />
[Bmmf(S)Ag].<br />
feeder layer of mouse peritoneal macrophages.<br />
B.malayi infective larvae (L3) obtained by the Positive clones were grown as ascites tumors in mice.<br />
dissection of infected mosquitoes were treated Purification of mAbs : Antibody producing cells<br />
similarly and the proteins obtained labeled as L3 (S) from isolated clones were propagated in vivo as ascites<br />
antigen. Brugia malayi adult worms were collected by in pristane- treated mice. SP2/0 myeloma cells were
JPD : Vol. 28 (1), 2004<br />
Monoclonal antibody Bm Ab120 in diagnosis of filariasis<br />
31<br />
injected similarly into mice <strong>for</strong> production of to check <strong>for</strong> cross-reactivity.<br />
irrelevant antibody <strong>for</strong> use in control experiments.<br />
Indirect ELISA: This technique was employed to<br />
Ascitic fluid was harvested from experimental and<br />
screen culture supernatant <strong>for</strong> antigen-specific<br />
control mice and centrifuged to remove cells.<br />
hybridomas and to check reactivity of mAb with<br />
Immunoglobulins (Ig) were isolated by ammonium different stages of B.malayi. Briefly, polystyrene<br />
sulphate precipitation as described earlier plates (Nunc) were coated with 100 micro litres (5<br />
(Subrahmanyam et al.,1990').<br />
micro grams /ml) of Bmmf (S)/ A(S) / L3(S) antigen in<br />
sodium carbonate-bicarbonate buffer(pH 9.6) and<br />
Polyclonal antibodies : Filarial serum<br />
incubated at 4° C overnight. Plates were drained and<br />
immunoglobulins (FSIgG ) from serum of clinical<br />
incubated in 0.5% gelatin in PBS <strong>for</strong> 1h at 37°C. Plates<br />
patients were isolated by ammonium sulphate<br />
were then washed in PBS-Tween 20 (PBS/T) once.<br />
precipitation followed by ion-exchange<br />
chromatography on DEAE column as described earlier One hundred micro litres of hybridoma supernatants /<br />
(Alikhan et al, 1990) while anti-Bmmf (S) antibodies Bm Abl20 were then added and incubated at 37°C <strong>for</strong><br />
were isolated from rabbits immunized with Bmmf (S) 1h. After 3 washes, wells were incubated with 100<br />
antigen similarly.<br />
micro litres of goat anti-mouse peroxidase conjugate<br />
and incubated <strong>for</strong> 1h at 37°C, washed 5 times and<br />
Human sera: Blood samples were collected from<br />
incubated with 100 micro litres of ABTS-H2O<br />
2<br />
villages surrounding Sevagram, Maharashtra, India<br />
that are highly endemic <strong>for</strong> bancroftian filariasis and<br />
substrate. <strong>The</strong> colour developed was read at 415nm.<br />
from patients visiting the out-patient department of Sandwich ELISA: This was done to screen sera of<br />
Kasturba Hospital, Sevagram with clinical symptoms endemic population with monoclonal antibody <strong>for</strong><br />
of filariasis such as lymphoedema, hydrocele, specific antigen.. Sandwich ELISA was per<strong>for</strong>med by<br />
elephantiasis, lymphadenitis and epididymoorchitis. coating 5 micro grams /ml of anti-Bmmf (S) or FSIgG<br />
<strong>The</strong> patients were classified into Grades I, II and III antibodies per well and incubating overnight at 4°C.<br />
depending upon their clinical symptoms as described Plates were washed once with PBS/T and blocked in<br />
earlier (Alikhan et al., 1990). Blood samples were also 1% BSA (Sigma) <strong>for</strong> 1 h at 37°C. Plates were washed<br />
collected from patients suffering from atypical again twice and incubated with 100 micro litres serum<br />
manifestations of filariasis such as Tropical (1:100 in PBS/T) <strong>for</strong> 1h at 37°C. After 5 washes, plates<br />
Pulmonary Eosinophilia (TPE), Urticaria and were incubated with 100 micro litres monoclonal<br />
bronchial asthma of suspected filarial etiology and antibody (1 micro gram/ml) <strong>for</strong> 1h at 37°C followed by<br />
grouped under 'Occult' filariasis. Twenty five of the 5 more washes and incubation with 100 micro litres of<br />
sera in this group comprised of TPE sera, 10 bronchial 1:2000 diluted goat anti-mouse HRPO <strong>for</strong> 1 h at 37°C.<br />
asthma cases and 5 cases of urticaria. Microfilaraemic Three mg of 2,2'- Azino- di (3-ethyl) benzthiazoline<br />
carriers were detected by presence of microfilariae on sulphonic acid (ABTS) in 10ml of citrate - phosphate<br />
microscopic examination of night blood smear. buffer (pH 5) and 4 micro litres of 30% H202<br />
was used as<br />
Asymptomatic, non-microfilaraemic individuals with substrate. <strong>The</strong> colour developed was read at 415 nm.<br />
no symptoms of the disease were considered endemic<br />
Western blot: Bmmf (S) antigens were separated on<br />
normals (EN). Non-endemic normal sera (NEN) were<br />
10% SDS-PAGE minigel under reducing conditions<br />
collected from Medical students admitted from nonalong<br />
with molecular weight markers. Antigens were<br />
endemic areas such as the states of Himachal Pradesh<br />
transferred to nitrocellulose paper (Hoefer) at 100 mA<br />
and Jammu & Kashmir. Ascaris, leishmania, malaria<br />
<strong>for</strong> 1 h and blocked with 4% skimmed milk powder <strong>for</strong><br />
and hookworm sera from non-endemic areas (a kind<br />
1 h at room temperature (RT). Supernatants from the<br />
gift from Sarika Gupta and Dr. Sushma Rathour,<br />
positive wells as well as ascitic fluid of the clones<br />
Banaras Hindu University) were also screened in order<br />
(1:5000 dilution) were added and incubated at room
32<br />
Monoclonal antibody Bm Ab120 in diagnosis of filariasis<br />
JPD : Vol. 28 (1), 2004<br />
temperature overnight. After washing 5 times with upon high ELISA titres.<br />
PBS/T anti-mouse horseradish peroxidase (1:2000)<br />
Properties of mAb; This monoclonal antibody was of<br />
was added and strips incubated <strong>for</strong> 1h. This was<br />
the IgM type as shown by isotyping by ELISA.<br />
followed by 5 washes and bands were developed by<br />
Immunoblot revealed that the mAb specifically<br />
adding diaminobenzidine/H2O 2substrate.<br />
reacted with a 120kDa antigen of Bmmf (S) as shown<br />
Isotyping: Ig isotypes produced by hybridomas were in Fig.l. <strong>The</strong> mAb will hence<strong>for</strong>th be referred to as Bm<br />
determined by using mAb-based mouse Ig isotyping Ab 120. <strong>The</strong> ability of this mAb to detect antigens from<br />
kit (Pharmingen).<br />
different stages of the parasite life cycle is shown in<br />
Fig.2. Bm Ab 120 showed high reactivity with Bmmf<br />
Ethical considerations: All animal experiments were<br />
(S) and A(S) antigens but low reactivity with L3 (S)<br />
carried out with prior permission from the Institute<br />
antigens of B.malayi.<br />
Animal Ethics Committee (IAEC). Blood samples<br />
were collected from microfilarial carriers during Screening of endemic population using Bm Ab 120:<br />
routine survey of the Public Health Department, Govt. IgM antibodies purified from ascitic fluid were used as<br />
of Maharashtra, after in<strong>for</strong>med consent.<br />
RESULTS<br />
Selection of monoclonal antibodies: Two out of 96<br />
hybrids showed the presence of antibodies towards the<br />
antigenic determinants present in whole Bmmf (S)<br />
antigen. <strong>The</strong>se two hybrids were cloned further to<br />
obtain several mAbs, of which one was selected based<br />
120 kDa<br />
TABLE : Detection of filarial antigen in different groups<br />
of sera using Bm Ab120 as probe in Sandwich ELISA<br />
Type of sera No. of sera No. positive (%)<br />
Screened FSIgG- Anti-Bmmf(S)-<br />
BmAb120 BmAb120<br />
Mf carriers 50 40(80) 44(88)<br />
Acute 10 8(80) 8(80)<br />
Grade I CP 15 11(73) 10(67)<br />
Grade II CP 15 9(60) 8(53)<br />
Grade III CP 10 5(50) 3(30)<br />
'Occult' 40 14(35) 10(25)<br />
EN 50 9(18) 6(12)<br />
NEN 15 0(0) 0(0)<br />
Mf carriers = Microfilaraemic carriers<br />
CP = Chronic pathology,<br />
EN = Endemic normals<br />
NEN = Non-endemic normals.<br />
Absorbance values of Mean + 2SD of the 50 EN screened<br />
was determined as the cut off value to distinguished between<br />
EN and infected individuals.<br />
97.4 kDa<br />
66 kDa<br />
45 kDa<br />
29 kDa<br />
Fig. 1. Immunoblot showing recognition of 120kDa component of<br />
Bm mf(S) by Bm Ab120.
JPD : Vol. 28 (1), 2004<br />
Monoclonal antibody Bm Ab120 in diagnosis of filariasis<br />
33<br />
OD VALUES AT 490 nm<br />
Absorbance<br />
Absorbance<br />
1<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
2<br />
1.6<br />
1.2<br />
0.8<br />
0.4<br />
0<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
1000 4000 8000 16,000<br />
RECIPROCAL TITRES OF BmAb 120<br />
Bm mf ES Bm A S Bm L3 S Bm mf S<br />
Fig. 2. Reactivity by ELISA of the different filarial antigens with the<br />
monoclonal antibody BmAb 120 (5 µg/ml protein).<br />
MFC CP OC EN NEN<br />
Sera screened<br />
Fig. 3. Result shown are Mean + SD obtained with sera diluted 1:100.<br />
MF = mf carriers, CP = clinical pathology, OC = suspected occult<br />
filariasis, EN = endemic normals, NEN = non-endemic normals.<br />
MFC CP OC EN NEN<br />
Fig. 4. Results shown are Mean + SD obtained with sera diluted 1:100.<br />
MF = mf carriers, CP = clinical pathology, OC = suspected occult<br />
filariasis, EN = endemic normals, NEN = non-endemic normals.<br />
a probe in a sandwich ELISA using two polyclonal<br />
antibodies [FSIgG and anti-Bmmf (S)] as capture<br />
antibodies. <strong>The</strong> results are summarized in the<br />
Table. <strong>The</strong> OD values <strong>for</strong> circulating filarial antigen<br />
using anti- Bmmf (S) antibodies were observed to be<br />
higher in all groups of sera compared to use of FSIgG<br />
(Figs.3 & 4). Absorbance values of Mean + 2SD of the<br />
50 EN screened was determined as the cut off value to<br />
distinguish between EN and infected individuals. With<br />
FSIgG as capture antibody, the assay was able to detect<br />
40/50 microfilaraemic carriers and 33/50 clinical<br />
cases. Nine of 50 endemic normals also had antigen<br />
levels beyond the cut-off. When anti-Bmmf (S)<br />
antibodies were used to capture filarial antigens, four<br />
more mf carriers were detected while the number of<br />
clinical cases detected was fewer by four. Only 6 EN<br />
were picked up as positive by this assay. Greater<br />
numbers of acute and early chronic cases were picked<br />
up as compared to Grade III stage of disease by both<br />
assays. Among the sera of probable occult filariasis<br />
screened, only 35% and 25% were above the cut-off<br />
titres of which TPE constituted the majority of cases.<br />
None of the fourteen non-endemic normals sera or sera<br />
of other parasitic infections screened were positive by<br />
either combination.<br />
DISCUSSION<br />
Various filarial antigens have been implicated in<br />
protective, pathogenic and allergic reactions in the<br />
host. Identification of specific filarial antigens would<br />
facilitate development of methods <strong>for</strong> detection of<br />
established infection. Several monoclonal antibodies<br />
have been developed against filarial antigens of which<br />
some are species and stage specific (Burkot et al.,<br />
1996) while others cross-react with epitopes of other<br />
nematodes (Subrahmanyam et al., 1990; Zheng et al.,<br />
1987). Some monoclonal antibodies are of diagnostic<br />
utility (Weil et al., 1987; More & Copeman, 1990)<br />
while some others have been used to confer passive<br />
immunity to the host (Parab et al., 1988). Sandwich<br />
ELISA using polyclonal and monoclonal antibodies<br />
against recombinant filarial antigens have also been<br />
used <strong>for</strong> screening of filarial sera (<strong>The</strong>odore & Kaliraj,<br />
1996). Two commercially available kits employing<br />
monoclonal antibodies directed against animal filarial
34 Monoclonal antibody Bm Ab120 in diagnosis of filariasis JPD : Vol. 28 (1), 2004<br />
parasites that have been widely used in<br />
epidemiological studies in filariasis include the rapid<br />
<strong>for</strong>mat test kit developed by ICT Diagnostics<br />
(AMRAD, Australia) and the Trop Bio Test. Various<br />
studies have confirmed the usefulness of these kits in<br />
detecting mf carriers with high sensitivity in different<br />
geographical areas (Bhumiratana et al, 1999;<br />
Simonsen et al, 1999). However, epidemiological<br />
studies have revealed that these tests show poor<br />
sensitivity in detecting amicrofilaraemic individuals<br />
with pathology and adult worm burdens (Nguyen et al,<br />
1999). <strong>The</strong> clinical manifestations in lymphatic<br />
filariasis range from acute symptoms (lymphadenitis,<br />
fever and chills), chronic presentations (elephantiasis<br />
and hydrocele) and occult manifestation (TPE,<br />
bronchial asthma, arthritis etc.). Most of the above<br />
individuals do not show the presence of mf in<br />
peripheral blood and hence may escape detection by<br />
ICT and Og4C3 assays that show high sensitivity in<br />
detecting microfilarial carriers. It is very difficult to<br />
diagnose atypical filarial manifestations and advise<br />
optimal DEC treatment <strong>for</strong> clinical relief and cure.<br />
Diagnosis offilaria etiology and morbidity control is<br />
also important in filaria control programme. Our<br />
studies with ICT have revealed 100% sensitivity in<br />
detecting mf carriers while only 1 of 6 clinical filarial<br />
cases was positive <strong>for</strong> antigen (Alli et al, 2001).<br />
Though ultrasonography techniques are available to<br />
screen amicrofilaraemic individuals carrying adult<br />
worms (Amaral et al, 7994), it is not a viable<br />
alternative to screen large populations. This<br />
necessitates the need to develop specific agents to<br />
detect antigenemia in not only mf carriers but also<br />
across a clinical spectrum of filariasis including those<br />
harbouring occult infections.<br />
Microfilariae in peripheral circulation of the host are<br />
picked by the mosquito vector where they undergo<br />
further development into the infectious L3 stage.<br />
Different circulating antigens from the microfilarial<br />
stage of the parasite have shown diagnostic potential<br />
(Balaji Ganesh et al, 2001; Alli et al, 2000,<br />
Chenthamarakshan et al, 1996). Development of<br />
mAbs against mf antigens could there<strong>for</strong>e facilitate the<br />
identification of such antigens. <strong>The</strong>re<strong>for</strong>e mAbs were<br />
generated using Bmmf (S) extract and the resulting<br />
mAbs used to screen a spectrum of population in an<br />
endemic area.<br />
Bm Ab 120 was of the IgM isotype and specifically<br />
recognized a 120 kDa fraction of B. malayi mf (S)<br />
antigen. <strong>The</strong> mAb also reacted with Bm mf ES antigen,<br />
BmA antigen and Bmmf ES antigen but poorly with<br />
BmL3 antigen. This is probably because the 120 kDa<br />
antigen is expressed highly in the mammalian stage of<br />
the parasite life cycle and less in the mosquito vector.<br />
Immunofluoresence studies revealed that the antigen<br />
was localized on the surface of the mf and adult stages<br />
of the parasite and not the L3 stage (data not shown).<br />
Circulating filarial antigen recognized by Bm Ab 120,<br />
thus, seems to be present in the mammalian stages of<br />
the filarial parasite life cycle.<br />
BmAbl20 was used as a probe in a sandwich ELISA<br />
using two different polyclonal antibodies, FSIgG and<br />
anti-Bm mf(S) as capture antibodies. FSIgG -Bm Ab<br />
120 could detect specific antigen in 80% mf carriers,<br />
66% of clinical filariasis and also picked up 18% of EN<br />
while anti Bmmf(S)- Bm Ab 120 sandwich ELISA<br />
could detect 88%, 58% and 12% of mf carriers,<br />
symptomatic cases and EN respectively. <strong>The</strong> increased<br />
sensitivity of the latter in detecting microfilarial<br />
carriers may be due to increased specificity of specific<br />
antibody in anti-Bmmf (S) serum as compared to<br />
FSIgG isolated from filarial patient sera. <strong>The</strong><br />
increased number of positives among the endemic<br />
normals with FSIgG resulted in lower difference in<br />
mean OD values between filarial and endemic normal<br />
groups as compared to the anti-Bmmf (S)- Bm Ab 120<br />
assay where mean OD of mf carriers was 3 times more<br />
than the endemic normals screened. <strong>The</strong> 80-88%<br />
sensitivity of the assays in detecting mf carriers in this<br />
study was lower than those reported earlier <strong>for</strong> the ICT<br />
or Trop Bio tests (90- 100%). However, a significantly<br />
high number of clinical cases were detected by this<br />
assay. <strong>The</strong> fact that many of the clinical cases, even<br />
though absent <strong>for</strong> microfilaraemia, showed presence<br />
of antigen was probably indicates the presence of<br />
active infecton. <strong>The</strong> assays could also detect filarial<br />
specific antigen in 25-35% of 'occult' cases, most of<br />
them TPE cases and 2 cases of bronchial asthma,
JPD : Vol. 28 (1), 2004<br />
Monoclonal antibody Bm Ab120 in diagnosis of filariasis<br />
35<br />
suggesting filarial etiology further emphasizing the Clin. Biochem.: 16 (2),207-10<br />
need to rule out filariasis in individuals with such Alli R, Bhandari YP, Reddy MVR and Harinath BC. 2000.<br />
atypical manifestations, particularly in children. Seroreactivity of purified Brugia malayi microfilarial soluble<br />
and excretory-secretory antigens in different clinical<br />
Antigen was not detected in pooled sera from few<br />
presentation of bancroftian filariasis. Ind. J. Exp. Biol, 38, 791-<br />
patients from non-endemic areas suffering from<br />
96.<br />
infections with hookworm, ascaris, malaria and<br />
Amaral F, Dreyer G, Figueredo-Silva J, Noroes J, Cavalcanti A,<br />
leishmania showing the monoclonal antibody is Samico SC, Santos A and Coutinho A. 1994. Live adult worms<br />
specific to filarial antigen. Screening of malayan sera detected by ultrasonography in human Bancroftian filariasis.<br />
could also reveal whether Bm Ab 120 is useful in Am J Trop Med Hyg.: 50 (6),753-7<br />
detecting B.malayi infected individuals as the mAb Balaji Ganesh B, Kader AM, Agarwal GS, Reddy MVR and<br />
reacts with both B.malayi mf and adult worms. Twelve Harinath BC. 2001. A simple and inexpensive dot-blot assay<br />
and eighteen percent of the endemic normals screened using a 66-kDa Brugia malayi microfilarial protein antigen, <strong>for</strong><br />
diagnosis of bancroftian filarial infection in an endemic area,<br />
were positive by the assays using different polyclonal<br />
Trans. Roy. Soc. Trop. Med. Hyg. 95, 168-169.<br />
antibodies. This is in agreement with some of the<br />
Bhumiratana A, Koyadun S, Suvannadabba S, Kamjanopas K,<br />
studies using ICT that have shown 14%- 24.7% of<br />
Rojanapremsuk J, Buddhirakkul P and Tantiwattanasup W.<br />
endemic normals as antigenemia positive 1999. Field trial of the ICT filariasis <strong>for</strong> diagnosis of<br />
(Bhumiratana et al, 1999. Njenga et al, 2001). Many Wuchereria bancrofti infections in an endemic population of<br />
endemic 'normals' are believed to harbour pre-patent Thailand. Southeast Asian J Trop Med Public Health : 30<br />
or cryptic infections or have undetectable levels of<br />
(3),562-8.<br />
microfilaraemia but show no overt clinical symptoms Braga C, Dourado MI, Ximenes RA, Alves L, Brayner F, Rocha A<br />
and Alexander N. 2003. Field evaluation of the whole blood<br />
resulting in considerable background positives. We<br />
immunochromatographic test <strong>for</strong> rapidbancroftian filariasis<br />
conclude that Bm Ab 120, when used with other diagnosis in the northeast of Brazil. Epub May-Jun; 45 (3), 125-<br />
suitable polyclonal antibodies may help in the 9.<br />
detection of circulating filarial antigen in Burkot T R, Kwan -Lim GE & Maizels RM. 1996. A novel 95-<br />
identification of infected individuals living in an kilodalton antigen of Wuchereria bancrofti infective larvae<br />
endemic area. Further multicentric studies are identified by species-specific monoclonal antibodies, Infect.<br />
required <strong>for</strong> strengthening the evidence base and <strong>for</strong><br />
Immun. 64 (2), 485-488.<br />
deciding the possible practical implication in Chandrasena TG, Premaratna R, Abeyewickrema W and de Silva<br />
NR. 2002. Evaluation of the ICT whole-blood antigen card test<br />
individual and community diagnosis of filariasis.<br />
to detect infection due to Wuchereria bancrofti in Sri Lanka.<br />
ACKNOWLEDGEMENTS<br />
Trans R Soc Trop Med Hyg : 96 (1), 60-3.<br />
Chenthamarakshan V, Reddy MVR and Harinath BC. 1996.<br />
This work was supported by the Department of<br />
Diagnostic potential of fractionated Brugia malayi<br />
Biotechnology, New Delhi. <strong>The</strong> authors are grateful to microfilarial excretory /secretory antigen <strong>for</strong> bancroftian<br />
Shri. Dhirubhai Mehta , President of Kasturba Health filariasis. Trans. Roy. Soc. Trop. Med. Hyg. 90 : 252-254<br />
<strong>Society</strong>, Dr. P. Narang, Dean, MGIMS and Dr. G.C. Eigege A, Richards FO Jr, Blaney DD, Miri ES, Gontor I, Ogah G,<br />
Mishra, Director, NCCS <strong>for</strong> their keen interest and Umaru J, Jinadu MY, Mathai W, Amadiegwu S and Hopkins<br />
encouragement in this work.<br />
DR. 2003. Rapid assessment <strong>for</strong> lymphatic filariasis in central<br />
Nigeria: a comparison of the immunochromatographic card test<br />
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Journal of Parasitic Diseases<br />
Vol.28 (1) June 2004, pp. 37-44<br />
Anthelmintic efficacy of extract of Stephania glabra and<br />
aerial root extract of Trichosanthes multiloba in vitro : two<br />
indigenous plants in Shillong, India<br />
1<br />
V TANDON*, LM LYNDEM, PK KAR, P PAL, B DAS, HSP RAO<br />
Department of Zoology, North Eastern Hill University, Shillong-793022, India<br />
1<br />
Department of Chemistry , Pondicherry University, Pondicherry, India.<br />
In the traditional medicinal practices in Meghalaya (Northeast India) aqueous concoction of the powders of<br />
the dried rhizome of Stephania glabra and aerial root of Trichosanthes multiloba is used as anthelmintic<br />
against intestinal worms. To ascertain their efficacy, the alcoholic crude extracts of these materials were<br />
tested against the various helminth parasites - nematode: Heterakis gallinarum, Ascaridia galli, Ancylostoma<br />
ceylanicum and Ascaris suum; cestode: Raillietina echinobothrida and trematode: Fasciolopsis buski in<br />
dosages ranging between 25-100 mg/ml in 0.9% phosphate buffered saline (PBS, pH 7.2) at 38 ± 1°C. <strong>The</strong><br />
controls, kept in PBS, survived <strong>for</strong> average 22 h (trematode), 72 h (cestode) and 55->380 h (nematodes).<br />
Treatment with S. glabra showed pronounced effect on the cestode and trematode; a dose-dependent<br />
gradual decline in physical motility was observed in R. echinobothrida and F. buski. <strong>The</strong> isolated active<br />
principle (Compound X) of the rhizome pulp also showed good efficacy against F. buski. In contrast, much<br />
less effect was revealed in respect of nematodes; in treatments with 50 mg/ml all the smaller nematodes<br />
attained paralysis within 7-10 h, whereas A. suum remained unparalyzed even after 40 h. In treatments with<br />
Trichosanthes alone the onset of paralysis in all the test worms took much longer than Stephania, though in<br />
concoction of the two crude extracts the paralytic effect was discernible in less than half the time taken <strong>for</strong><br />
individual treatments. <strong>The</strong> phytochemical(s) of S. glabra seems to be effective against platyhelminth<br />
parasites that have a tegumental interface, but not so against cuticle-covered nematodes.<br />
Keywords: Anthelmintic; Cestode; Nematode; Parasites; Stephania glabra; Trichosanthes multiloba;<br />
Trematode<br />
A<br />
large number of plants with medicinal<br />
properties have been reported from India<br />
(Chopra et al., 1956; Dhar et al., 1965, 1968, 1973;<br />
Dhawan et al., 1980; Rao and Krishnaiah, 1982;<br />
Bhakuni et al., 1988; Shilaskar and Parasher, 1989)<br />
and some of them have been documented as curative<br />
against worm infections (Perry, 1980; Dharma, 1985;<br />
Chhabra et al., 1990). Meghalaya, one of the<br />
biodiversity hotspots of Northeast India, has a wide<br />
variety of plants used by the natives as curatives<br />
against worm infections (Rao, 1981). Besides<br />
* Corresponding Author<br />
Flemingia vestita (family Fabaceae) that is used<br />
widely as an anthelmintic against gastrointestinal<br />
worms (Tandon et al., 1997), two other plants,<br />
Stephania glabra Miers (family Menispermaceae) and<br />
Trichosanthes multiloba Clarke (family<br />
Cucurbitaceae), are similarly used in the traditional<br />
medicinal system. While the rhizome pulp of S. glabra<br />
is used as anthelmintic locally, the aerial root of T.<br />
multiloba is used in concoction with S. glabra pulp <strong>for</strong><br />
more effective cure.<br />
With a view to establishing anthelmintic efficacy of<br />
the above-mentioned plants and to find out clues to the<br />
plausible mode of their action, a thorough study
38 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004<br />
seemed desirable. <strong>The</strong> present study, there<strong>for</strong>e, aimed using about 300 ml of mixed solvent at each ratio.<br />
at investigating the efficacy of the phytochemicals on<br />
From the rhizome pulp and peel only one major<br />
the model nematode, cestode and trematode parasites<br />
isoflavone could be isolated, the chemical constitution<br />
of zoonotic significance. Physical motility and of which is yet to be ascertained; hereafter this is<br />
survival time including the onset of the paralytic state<br />
referred to as Compound X. For T. multiloba, due to<br />
and alterations in the surface architecture of the<br />
very less quantity of the crude extract recovered from<br />
parasites constitute the parameters of activity.<br />
the available material, isolation of active components<br />
MATERIALS AND METHODS<br />
could not be pursued.<br />
Plant crude extract: Fresh rhizomes of S. glabra and Parasites: Parasites (nematodes: Heterakis<br />
aerial roots of T. multiloha were collected in winter gallinarum and Ascaridia galli from domestic fowl<br />
from Mawsynram, a small hamlet, about 60 km from and Ascaris suum from pigs; cestode: Raillietina<br />
Shillong (Meghalaya). After washing thoroughly with echinobothrida from domestic fowl; trematode:<br />
water the rhizomes were thinly peeled and the pulp was Fasciolopsis buski from pigs) were collected in<br />
diced into small pieces; the aerial roots were also phosphate buffered saline (PBS; pH 7.2) from freshly<br />
chopped into small unpeeled pieces. Each of these slaughtered hosts at the local abattoirs. Ancylostoma<br />
materials was soaked separately in rectified alcohol ceylanicum was obtained from infected hamsters<br />
<strong>for</strong> about a month; in the case of S. glabra, the maintained in the laboratory. <strong>The</strong> parasites were<br />
alcoholic contents turned dark brown, whereas the exposed to different treatments within 1 h of<br />
solvent with T. multiloba aerial roots attained only a collection.<br />
golden tinge. <strong>The</strong> alcoholic solutions thus obtained<br />
Treatments: <strong>The</strong> test parasites were incubated at 38 ±<br />
were distilled in a reflux condenser <strong>for</strong> isolating the<br />
1°C in 0.9% phosphate buffered saline (PBS, pH 7.2)<br />
concentrated crude extract.<br />
containing crude extract in concentrations of 25, 50<br />
Finally 200 g dry crude extract was obtained from 4 kg and 100 mg/ml supplemented with 1 % DMSO. While<br />
pulp, and 40 g from 2.5 kg peel of S. glabra. From F. buski and A. suum were kept singly <strong>for</strong> each test, H.<br />
about 5 kg aerial roots of T. multioloba, 50 g crude gallinarum and A. ceylanicum were incubated in<br />
extract was retrieved.<br />
batches of 5 worms, each of approximately same size.<br />
For R. echeinobothrida, on the other hand, 5 strobila,<br />
Compound X: Purified active component from the<br />
each with scolex and of approximately equal length,<br />
crude extract of S. glabra rhizome pulp and peel was<br />
were used. For each experiment three replicates were<br />
isolated following the method of Rao and Reddy<br />
used. <strong>The</strong> reference drugs praziquantel (PZQ,<br />
(1991). <strong>The</strong> alcoholic crude extract of the plant<br />
cestocide), oxyclozanide (flukicide) and mebendazole<br />
material was mixed well with hexane and the<br />
(nematocide) were used in various concentrations with<br />
supernatant solution was decanted. This process was<br />
1 % DMSO, as established in earlier studies (Visen et<br />
repeated 20 times. <strong>The</strong> extract was reduced in volume<br />
al., 1987; Roy and Tandon, 1996; Tandon et al., 1997).<br />
through distillation and passed through silica gel<br />
Controls were maintained in 0.9 % PBS with 1%<br />
column using hexane, benzene and ethyl acetate as<br />
DMSO at 38 ± 1°C. <strong>The</strong> parasites, both in control and<br />
solvents. <strong>The</strong> column was prepared using hexane and<br />
treated, were brought back to the slightly warm PBS to<br />
benzene mixture (6:4) and silica gel 100-200 mesh<br />
examine their motility at every 15 min gap and the time<br />
(about 350 g). <strong>The</strong> sample (10 g) was mixed with silica<br />
taken <strong>for</strong> the reversible loss of motility (paralysis) and<br />
gel 60-120 mesh and loaded on top of the column.<br />
death (no motility) was recorded as per the procedure<br />
Elution was carried out with hexane: benzene mixture<br />
followed in earlier similar study (Tandon et al., 1997).<br />
successively at the ratio of 6:4, 5:5, 4:6 and so <strong>for</strong>th to<br />
pure benzene and thereafter with benzene: ethyl Scanning electron microscopy: Soon after they<br />
acetate (9:1, 8:2, 7:3, 6:4, 5:5 till pure ethyl acetate) attained a paralytic state, the test materials (R.
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of rhizome extract<br />
39<br />
echeinobothrida, F. buski and A. ceylanicum), treated high efficacy at a concentration of 1.0 mg/ml, with<br />
with various dosages of the rhizome-pulp extract of S. paralysis ensuing in less than 1 h.<br />
glabra, were fixed along with their respective controls<br />
<strong>The</strong> aerial root extract of T. multiloba showed a<br />
in 10% neutral buffered <strong>for</strong>malin at 4°C <strong>for</strong> 24 h,<br />
similar pattern of efficacy; at the concentration of<br />
washed in PBS and dehydrated with ascending grades<br />
l00 mg/ml, paralysis occurred at 2.19 ± 0.001 h,<br />
of acetone to pure dried acetone. <strong>The</strong> specimens were<br />
2.41 ± 0.52 h, 5.26 ± 0.072 h and 11.02 ± 0.003 h in<br />
then critical-point dried using liquid carbon dioxide as<br />
the case of R. echinobothrida, F. buski, A.<br />
the transitional fluid or treated with tetramethylsilane<br />
ceylanicum and A. galli, respectively. This efficacy<br />
following Roy and Tandon (1991). <strong>The</strong> gold-coated<br />
is more or less comparable to that of S. glabra.<br />
specimens were observed using LEO 435 VP SEM or<br />
However, in the concoction of the two plant<br />
JEOL at electron-accelerating voltages ranging materials (1:1) the time taken <strong>for</strong> paralysis in the<br />
between 10-20 kV.<br />
test parasites lowered significantly in comparison<br />
Statistical analysis: Data were statistically analyzed to the treatment with the crude-pulp extract of S.<br />
and presented as mean ± SEM (n = 3). Comparisons of glabra alone, indicating a synergistic effect of the<br />
the paired mean values between the experimental and two at a dosage of 50 or 100 mg/ml.<br />
respective reference were calculated using Student's<br />
All the nematode species showed a negligible effect as<br />
t-test (Croxton et al., 1982) and differences with<br />
they survived the various treatments <strong>for</strong> comparatively<br />
P
40 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004<br />
Figs. 1-6. Surface architecture of the parasites following treatment with<br />
S. glabra crude rhizome pulp extract (100 mg/ml):<br />
1, 2. Fasciolopsis buski- ventral tegument<br />
1. Control, showing the spination of the normal surface.<br />
(Scale bar = 10µm).<br />
2. Treated fluke; distortion of the spines, sloughing and<br />
pits are noticeable.<br />
(Scale bar = 30µm).<br />
3, 4. Ancylostoma ceylanicum- cuticle, mid body<br />
3. Control, showing striated surface pattern.<br />
(Scale bar = 2.5µm).<br />
4. Treated; not much distortion of the cuticular<br />
architecture is discernible even at higher<br />
magnification.<br />
(Scale bar = 5µm).<br />
5, 6. Raillietina echinobothrida- tegumental surface of the<br />
gravid proglottid<br />
5. Control, showing normal surface contour.<br />
(Scale bar =100 µm).<br />
6. Treated worm; cracks and wrinkles in the tegumental<br />
surface, with clumpy microtriches are evident.<br />
(Scale bar =100 µm).
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of rhizome extract<br />
41<br />
Table : Efficacy of the rhizome pulp and peel extracts of Stephania glabra and aerial root extract of Trichosanthes<br />
multiloba against various helminth parasites*<br />
Time (h) taken <strong>for</strong> complete loss of movement<br />
Treatment Raillietina Fasciolopsis Heterakis Ancylostoma Ascaridia Ascaris<br />
(mg/ml) echinobothrida buski gallinarum ceylanicum galli suum<br />
S. glabra<br />
Crude rhizome pulp extract<br />
25 7.4+0.05 5.04+0.02 - 11.38+0.12 - -<br />
a a b<br />
50 2.24+0.17 2.34+0.46 10.19+0.16 7.31+0.03 10.2+0.07 55.04+0.03<br />
a a b a b x<br />
100 1.44+0.11 0.91+0.21 7.19+0.09 4.20+0.05 6.9+0.04 43.54+0.01<br />
Crude rhizome peel extract<br />
25 - - - 11.01+0.02 - -<br />
b<br />
50 5.05+0.13 - - 6.80+0.07 - -<br />
b<br />
a<br />
100 3.01+0.05 - - 3.18+0.04 - -<br />
Compound X<br />
0.5 - 1.37+0.01 - - - -<br />
b<br />
1.0 - 0.74+0.02 - - - -<br />
T. multiloba<br />
Crude aerial root extract<br />
25 3.76+0.01 - - 12.6+0.1 15.58+0.01 -<br />
x b x<br />
50 3.39+0.02 4.07+0.71 - 8.1+0.02 13.7+0.01 -<br />
b b a b<br />
100 2.19+0.01 2.41+0.52 - 5.26+0.07 11.02+0.01 -<br />
S. glabra (pulp) : T. multiloba (1:1)<br />
50 - 1.18+0.03 - 3.21+0.04 - -<br />
a<br />
b<br />
100 - 0.32+0.05 - 2.71+0.04 - -<br />
Praziquantel<br />
0.001 2.9+0.05 - - - - -<br />
a<br />
0.005 0.89+0.04 - - - - -<br />
b<br />
0.01 0.47+0.07 - - - - -<br />
Oxyclozanide<br />
5 - 1.97+0.01 - - - -<br />
b<br />
10 - 1.12+0.02 - - - -<br />
a<br />
20 - 0.68+0.03 - - - -<br />
Mebendazole<br />
2.5 - - - 5.08+0.03 - -<br />
b<br />
5 - - - 3.78+0.02 - -<br />
a<br />
10 - - - 2.51+0.02 - -<br />
* <strong>The</strong> parasites survived in control (0.1% DMSO in PBS, pH 7.2) <strong>for</strong> considerably longer periods than in any<br />
treatments : R.echinobothria, 72+0.05 h; F. buski, 20+2 h; H. gallinarum, 120.04+0.04 h; A. ceylanicum, 56.5+0.05<br />
h; A. galli, 168+0.08 h and A. suum, 384+0.1 h. Values are expressed as mean + S.E.M. (n=3).<br />
- not tested.<br />
a, b<br />
: p-value significant at < 0.01 and < 0.05 level, respectively in comparison to lower dosages.<br />
x<br />
: Not significant.
42 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004<br />
DISCUSSION<br />
treatment with PZQ, are well established<br />
(Mehlhorn et al., 1983; Schmahl and Mehlhorn,<br />
<strong>The</strong> present study demonstrates that the test plant<br />
1985). Tegumental bubbles of different sizes<br />
materials have a deleterious effect on R.<br />
appeared on the surface of Opisthorchis viverrini<br />
echinobothrida and F. buski. In ultrastructural studies,<br />
treated with PZQ in vitro and in vivo. Thus these<br />
disorientation of the microtriches, spines and scales<br />
ultrastructural changes may represent a generalized<br />
was observed along with severe distortion and<br />
response of the tegumental surface to an obnoxious<br />
de<strong>for</strong>mity of the tegument, breakage and sloughing off<br />
agent (Sirisinha et al., 1984). <strong>The</strong> same fluke when<br />
of the tegumental surface structures. Cestodes and<br />
treated with amoscanate exhibited severe swelling<br />
trematodes are soft-bodied parasites and the tegument<br />
and pit <strong>for</strong>mation, leading to total disruption of the<br />
is their only or major interface, through which various<br />
surface tegument and suggested that the drug may<br />
basic physiological functions such as digestion and<br />
have caused an imbalance in osmosis, resulting in<br />
absorption take place. <strong>The</strong> plant-derived components<br />
impaired ion transfer (Sobhon et al., 1986). <strong>The</strong><br />
seem to affect this normal physiological functioning<br />
changes in the tegumental architecture on treatment<br />
causing paralysis and subsequent mortality of the<br />
with various test materials of plant origin suggest<br />
parasite, as has been described be<strong>for</strong>e in case of R.<br />
that the plant products bring about permeability<br />
echinbothrida and F. buski on being treated with F.<br />
changes in the tegument of the worm.<br />
vestita and its active component genistein (Roy and<br />
Tandon, 1996; Tandon et al., 1997; Pal and Tandon, Disruptions of the cuticular interface and/or<br />
1998). intestinal epithelium and degenerative changes<br />
even in the subcuticular region have been reported<br />
Tegumental distortion and severe vacuolization on<br />
in several nematode species exposed to<br />
exposure to flukicidal drugs have been observed in<br />
anthelmintics in vitro (An, 1990; Storte et al., 1990;<br />
several species of trematodes (Jiang and Xia, 1992;<br />
Mackenstedt et al., 1993; Rothwell and Sangster,<br />
Schmahl, 1993; Stitt and Fairweather, 1993; Xu-<br />
1996). Though there was also an orderly decrease in<br />
Lin et al., 1994; Anderson, and Fairweather, 1995);<br />
the time taken <strong>for</strong> paralysis with increasing<br />
the extent of damage induced was reported to<br />
concentration of the plant-derived components in<br />
increase with the exposure time. Similar changes<br />
the nematode species in the present study, the onset<br />
were noticed in case of cestode parasites (Delabreof<br />
paralysis in them took much longer time than the<br />
Defayolle et al., 1989; Perez et al., 1994; Pal and<br />
platyhelminth parasites. <strong>The</strong> synergistic effect of S.<br />
Tandon, 1998). In Fasciola hepatica, treatment<br />
glabra and T. multiloba concoction (1:1) at 50 or<br />
with diamphenetide caused blebbing of the 100 mg/ml showed more efficacious results<br />
tegument and the blebs increased in size with an<br />
compared to the effect in treatments with individual<br />
increase in the dosage; surface pitting was also<br />
plant materials.<br />
observed and the mid body region of the tegument<br />
was stripped off to expose the basal lamina beneath It can be hypothesized that the paralysis of the flat-<br />
(Fairweather et al., 1987). <strong>The</strong> ultrastructural bodied helminth parasites on exposure to the plantchanges<br />
in the tegument are linked to a possible derived components is attributable to the severe<br />
mode of action of the drug as an inhibitor of protein distortion, degeneration and disruption of the<br />
synthesis (Anderson and Fairweather, 1995). tegumental architecture of the parasite. <strong>The</strong> plant-<br />
Vacuolization and contraction in the parasite body derived components do not seem to be much<br />
2+<br />
surface are closely related to the levels of Ca effective against the cuticle-covered nematodes. It<br />
concentration of the media used (Bricker et al., may further be assumed that these phytochemicals<br />
1982; Xiao et al., 1984). Disturbances in ion flux have a vermifugal action in that the paralyzed<br />
across the membrane, leading to changes in the parasites are removed from the host by peristaltic<br />
tegumental integrity in different trematodes on movements of the intestine.
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of rhizome extract<br />
43<br />
ACKNOWLEDGEMENTS<br />
Carica papaya seeds. <strong>Indian</strong> J. Pharmacol. 27:335-336.<br />
Dharma AP. 1985. Tanaman obat traditional Indonesia, Jakarta,<br />
This work was supported by GBPIHED (Ministry of<br />
PN. Balai Pusterka.<br />
Environment & Forests, GOI)-sponsored project to<br />
Dhawan BN, Dubey MP, Mehrotra BN, Rastogi RP and Tandon JS.<br />
VT and DRS-III project of the University Grants<br />
1980. Screening of <strong>Indian</strong> plants <strong>for</strong> biological activity: Part IX.<br />
Commission, GOI, in the Department of Zoology, <strong>Indian</strong> J. Exp. Biol. 118(6): 594-606.<br />
North Eastern Hill University, Shillong. Thanks are<br />
Fairweather I, Anderson HR and Baldwin TM. 1987. Fasciola<br />
also due to the Head, Department of Chemistry, hepatica. tegumental surface alterations following treatment in<br />
Pondicherry University, <strong>for</strong> allowing the usage of vitro with deacetylated (amine) metabolite of diamphencthide.<br />
laboratory facility <strong>for</strong> extraction of active components Parasitol. Res. 73(2):99-106.<br />
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A.I.I.M.S., New Delhi and Director, R.S.I.C., NEHU, observation on the effects of praziquantel and albendazole on<br />
Paragonimus heterotremus in rats. Chin. J. Parasitic Dis. Contr.<br />
Shillong <strong>for</strong> SEM facility.<br />
5(4):264-266.<br />
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Dhar RN, Garg LC and Pathak RD. 1965. Anthelmintic activity of<br />
Roy B and Tandon V. 1991. Usefulness of tetramethylsilane in the
44 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004<br />
preparation of helminth parasites <strong>for</strong> scanning electron Stitt AW and Fairweather I. 1993. Fasciola hepatica: tegumental<br />
microscopy. Rivista di Parasitologia 8(3) : 207-215.<br />
surface changes in adult and juvenile flukes following<br />
treatment in vitro with the sulphoxide metabolite of<br />
Schmahl G. 1993. Treatment of fish parasites. 10. Effects of a new<br />
triclabendazole (Fasinex). Parasitol. Res. 79(7):529-536.<br />
triazine derivative HOE 092V, on Monogenea: a light and<br />
transmission electron microscopy study. Parasitol. Res. Storte G, Darge K and Bonow I. 1990. Morphological alterations of<br />
79:559-566. male Onchocerca volvulus after in vitro exposure to Mel W and<br />
Milbemycin A confirming the results of viability tests. Trop. J.<br />
Schmahl G and Mehlhorm H. 1985. Treatment of fish parasites. 1.<br />
Med. Parasitol. 41:429-436.<br />
Praziquantel effective against monogenea (Dactylogyrus<br />
vastator, Dactylogyrus extensus, Diplozoon paradoxum). Z. Tandon V, Pal P, Roy B, Rao HSP and Reddy KS. 1997. In vitro<br />
Parasitenkde. 71:727-737.<br />
anthelmintic activity of root tuber extract of Flemingia vestita,<br />
an indigenous plant in Shillong, India. Parasitol. Res. 83:492-<br />
Shilaskar DV and Parasher GC. 1989. Evolution of indigenous<br />
498.<br />
anthelmintics. In vitro screening of some indigenous plants <strong>for</strong><br />
their anthelmintic activity against Ascaridia galli. <strong>Indian</strong> J. Visen PKS, Mishra A and Katiyar JC. 1987. Speed action of methyl<br />
Indigenous Med. 6: 49-53.<br />
5(6)-[4-(2-pyridyl)] piperazinocarbamoylbenzimidazole-2-<br />
carbamate, mebendazole and thiabendazole against<br />
Sirisinha S, Puengtomwatanakul S, Sobhon P, Saitongdee P,<br />
experimental hookworm infections. <strong>Indian</strong> J. Exp. Biol.<br />
Wongpayabal P, Mitranonde V, Radomyos P, Bunnag D and<br />
25:695-699.<br />
Harinasuta T. 1984. Alterations of the surface tegument of<br />
Opisthorchis viverrini exposed to praziquantel in vitro and in Xiao S, Friedman PA, Catto BA, Webster LT Jr. 1984. Praziquantel<br />
vivo. Southeast Asian J. Trop. Med. Public Health 15(1): 95- - Induced vesicle <strong>for</strong>mation in the tegument of male<br />
103. Schistosoma mansoni is calcium dependent. J. Parasitol.<br />
Sobhon P, Wanichanond C, Sattongdee P, Koonchornboon T,<br />
70:177-179.<br />
Bubphaniroj P, Upatham ES, Puengtomwatanakul S and Xu-Lin H, Zhou SJ, Lian WN, Mao MZ and Yu YF. 1994. Electron<br />
Sirisinha S. 1986. Scanning electron microscopic study of microscopic observations of tegumental damage in adult<br />
Opisthorchis viverrini tegument and its alterations induced by Schistosoma japonicum after in vivo treatment with<br />
amoscanate. Int. J. Parasitol. 16:19-26.<br />
levopraziquantel. Chin. Med. J. 107(10):771-774.
Journal of Parasitic Diseases<br />
Vol. 28 (1) June 2004, pp. 45-56<br />
Anthelmintic efficacy of genistein, the active principle of<br />
Flemingia vestita (Fabaceae): Alterations in the activity of the<br />
enzymes associated with the tegumental and gastrodemal<br />
interfaces of the trematode, Fasciolopsis buski<br />
PRADIP KUMAR KAR and VEENA TANDON*<br />
Department of Zoology, North-Eastern Hill University, Shillong - 793022<br />
Acid phosphatase (AcPase) and alkaline phosphatase (AlkPase) were found widely distributed in<br />
tegumental tissues, i.e., tegument, subtegument and somatic musculature as well as in the gastrodermis of<br />
the intestinal fluke Fasciolopsis buski. Adenosine triphosphatase (ATPase) and 5'-Nucleotidase (5'-Nu), to<br />
the contrary, were localized in the tegumental tissues only while protease was associated with the lattermentioned<br />
tissues. After exposure to the various test materials (crude root-peel extract of Flemingia vestita<br />
20 mg/ml, its active component genistein 0.5 mg/ml and the reference drug oxyclozanide 20 mg/ml<br />
concentration) a significant decline in the visible stain intensity of all these enzymes except protease was<br />
observed in the a<strong>for</strong>ementioned regions. Quantitatively, the activities of AcPase, AlkPase, ATPase, and 5'-<br />
Nu were found to diminish by 39-42.75%, 37.5-44.23%, 32.9-54.5% and 41.9-51.9%, respectively after<br />
treatment. <strong>The</strong> reference drug showed somewhat similar effect like the crude extract of F. vestita.<br />
Keywords: Acid phosphatase, Adenosine triphosphatase, Alkaline phosphatase, Anthelmintic, Flemingia<br />
vestita, Genistein, 5'-Nucleotidase, Phytochemicals, Protease, Tegumental enzymes, Trematode<br />
he presence of several enzymes viz., acid metacercarial cyst of Bolbogonotylus corkumi and<br />
Tphosphatase (AcPase), alkaline phosphatase adult Fibricola seoulensis, an intestinal trematode of<br />
(AlkPase), adenosine triphosphatase (ATPase) and 5'- human and rodents, showed moderate AcPase activity<br />
Nucleotidase (5'-Nu) has been detected both (Walker and Wittrock, 1992; Huh, 1993). <strong>The</strong><br />
histochemically and biochemically in a number of functional characteristics of the tegument and<br />
helminth parasites, wherein these enzymes have been intestinal wall were studied in mature Fasciola<br />
found to be closely associated with the tegument or hepatica after treatment with luxabendazole, and<br />
cuticle, subtegument, somatic musculature and gut alterations were noted in the activities of AcPase,<br />
(Kwak and Kim, 1996; Buchmann, 1998; Fetterer and ATPase, inosine triphosphatase and succinate<br />
Rhoads, 2000). AcPase and AlkPase occur widely in dehydrogenase (Gorchilova et.al., 1990). <strong>The</strong> aqueous<br />
the tegument of trematodes, cestodes and extract of some medicinal plants viz., Butea<br />
acanthocephalans. In adult cestodes AlkPase is usually monosperma, Embelia ribes and Roltlesia tinctoria<br />
the most active, whilst AcPase tends to predominate in caused reduction in both AcPase and AlkPase<br />
the trematode tegument (Barrett, 1981). <strong>The</strong> activities of Paramphistomum cervi in vitro (Chopra et<br />
al., 1991). Praziquantel (PZQ), on the other hand,<br />
* Corresponding Author<br />
induced AlkPase activity on the surface of the adult<br />
Schistosoma mansoni (Fallon et. al., 1994).
46 Anthelmintic efficacy of genistein<br />
JPD : Vol. 28 (1), 2004<br />
Majority of the tegumental enzymes are believed to be s e v e r a l h e l m i n t h s p e c i e s i n c l u d i n g t h e<br />
involved in digestion and/or absorptive function in liver/intestinal/blood/lung flukes, haematophagous<br />
cestodes (Poljakova et al., 1983). McCracken and and tissue dwelling nematodes, and cestodes (review-<br />
Taylor (1983) reported biochemical effects of Trap and Boireau, 2000). In view of the differences in<br />
thiabendazole and cambendazole on Hymenolepis the inhibition properties from their host<br />
diminuta in vivo; the failure of glucose uptake could counterparts and specific capacities <strong>for</strong><br />
lead to the depletion of parasite AlkPase activity. immunomodulation the parasite proteases are<br />
<strong>The</strong> AcPase activity in the isolated brush border currently being proposed as major immuno- and<br />
membrane of H. diminuta was inhibited chemotherapeutic targets (Hawthorne et al., 2000;<br />
particularly by levamisole, 2-mercaptoethanol and Hartmann and Lucius, 2003) and their potential in<br />
EDTA (Pappas, 1988).<br />
the development of vaccines is being exploited<br />
<strong>The</strong> broad spectrum anthelmintics parbendazole and<br />
(Dalton et al., 2003; Williams et al., 2003).<br />
piperazine adipate showed a marked reduction in the In a previous study, genistein, the active principle of<br />
activity of AcPase activity in the nematodes Ascaridia the root-peel of Flemingia vestita, a leguminous plant<br />
galli and Heterakis gallinae but the activities of lactate with edible tuberous roots, was shown to induce<br />
dehydrogenase and AlkPase were not affected alterations in the activity of tegumental enzymes in<br />
significantly (Sharma et al., 1987). Some drugs like cestodes (Pal and Tandon, 1998). In view of the<br />
phenothiazine, diethylcarbamazine and centperazine putative transtegumental functioning of this<br />
altered the metabolism and inhibited the <strong>for</strong>mation of phytochemical, we extended the study to trematode<br />
mitochondrial energy, thus depriving the parasite worms in order to ascertain if the plant-derived<br />
Setaria cervi of ATP (Agarwal et al., 1990). components influence the activity of enzymes<br />
Tetramisole and levamisole also caused inhibition of associated with the tegumental and gastrodermal<br />
++<br />
the specific activities of AcPase and Mg -dependent interfaces of these parasites.<br />
ATPase in A. galli, though AlkPase activity increased<br />
MATERIALS AND METHODS<br />
in the presence of both these drugs (Aggarwal et al.,<br />
1992). Thiabendazole and mebendazole changed the<br />
Parasites and test materials; Mature Fasciolopsis<br />
activity of AcPase in Nippostrongylus brasiliensis in<br />
buski were collected from the intestine of swine (Sus<br />
infected mice (Mishra and Srivastava, 1990).<br />
scrofa) obtained from the local abattoirs in 0.9%<br />
Rafoxamide also affected the key dehydrogenases<br />
Phosphate Buffered Saline (PBS, pH 7.2). <strong>The</strong> crude<br />
involved in the carbohydrate metabolism of Trichuris<br />
root-peel extract of F. vestita was prepared as<br />
globulosa by blocking the glycolytic pathway<br />
described earlier (Tandon et al., 1997). <strong>The</strong> parasites<br />
depriving the parasite of energy (Parveen et.al., 1992).<br />
were incubated with the crude extract (20 mg/ml),<br />
genistein (0.5 mg/ml) and oxyclozanide, the reference<br />
Proteases are known to play an important role in the<br />
drug (20 mg/ml) concentration (all prepared in 0.9%<br />
pathogenesis of several parasitic infections; besides<br />
PBS with 1% DMSO). <strong>The</strong> a<strong>for</strong>ementioned<br />
general catabolic functions, these proteolytic enzymes<br />
concentrations were chosen since at these dosages the<br />
may be significant in parasite immunoevasion, paralytic effect of the worm was attained within a<br />
excystment/evagination/encystment, and cell and shorter time frame as compared to lower<br />
tissue invasion (Park et al., 2001; Sajid and Mckerrow,<br />
concentrations (Roy and Tandon, 1996). Three<br />
2002). Proteases represent a very heterogeneous group<br />
replicates <strong>for</strong> each set of incubation medium were<br />
of proteins among parasites (Wijffels et al., 1994) and<br />
used. On attaining the paralytic state after<br />
have been isolated and characterized from the treatment the trematodes were further processed <strong>for</strong><br />
excretory/secretory products or tissue extracts of<br />
histochemical and biochemical studies, along with
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of genistein<br />
47<br />
containing moist filter paper. At 5 min intervals the<br />
sections were inspected to make sure that they were not<br />
too dry, in which case further buffer must be added.<br />
After incubation the preparation is dried without<br />
washing and subsequently dehydrated in alcohol,<br />
cleared in xylene and mounted in a synthetic medium.]<br />
<strong>The</strong> protease activity is shown as a clear area where the<br />
gelatin film and its contained granules have<br />
disappeared. Histological, but not intracellular,<br />
definition is obtained.<br />
Biochemical assays:<br />
AcPase and AlkPase : AcPase and AlkPase activities<br />
were assayed by estimating the p-nitrophenol product<br />
following the method as given by Plummer (1988)<br />
with necessary modification in the concentration of<br />
the buffer and substrate. One unit of AcPase or AlkPase<br />
activity is defined as that amount which catalyzed the<br />
<strong>for</strong>mation of 1 mM of p-nitrophenol/h at 37°C.<br />
ATPase: ATPase activity was assayed by estimating<br />
the free phosphate released, following the method of<br />
Kaplan (1957) with Na-ATP as the substrate. One unit<br />
of ATPase is defined as the amount which catalyzed the<br />
release of 1 µmole of phosphate / h at 37°C from ATP.<br />
5'-Nu: <strong>The</strong> enzyme activity was assayed by estimating<br />
the free phosphate released following the method of<br />
Bunitian (1970) using AMP as the substrate. One unit<br />
of 5'-Nu activity is defined as that amount which<br />
catalyzed the release of l µmole of phosphate/h at 37°C<br />
from AMP.<br />
Protein: <strong>The</strong> protein content was estimated following<br />
the method of Lowry et al. (1951) using bovine serum<br />
albumin as a standard.<br />
one set of control specimens (maintained in 1%<br />
DMSO in PBS).<br />
Histochemical localization:<br />
AcPase: <strong>The</strong> AcPase activity was demonstrated<br />
following the modified lead nitrate method of<br />
Takeuchi and Tanoue as described by Pearse (1968),<br />
wherein β-glycerophosphate was used as the substrate.<br />
A brownish precipitate indicates sites of AcPase.<br />
AlkPase: A modified coupling azo-dye method<br />
described by Pearse (1968) was used <strong>for</strong> the<br />
determination of AlkPase activity using Fast violet-B.<br />
<strong>The</strong> sites of AlkPase activity are coloured brown with<br />
salts, nuclei dark blue.<br />
ATPase: For the demonstration of ATPase activity,<br />
calcium method using Na-ATP as the substrate, after<br />
Maengwyn-Davies et al., as described by Pearse<br />
(1968) was followed. <strong>The</strong> activity of ATPase shows as<br />
a blackish brown deposit.<br />
5'-Nu: For the study of 5'-Nu activity the lead method<br />
after Wachstein and Meisel (1957) was employed<br />
using adenosine monophosphate (AMP) as the<br />
substrate. Yellow deposits of lead sulphide indicate<br />
sites of 5'-Nu enzyme activity.<br />
Protease: For the demonstration of protease the gelatin<br />
method of Adams and Tuqan (1961) as described by<br />
Pearse (1968) was followed.<br />
[<strong>The</strong> specimens were fixed in cold (4°C) 4%<br />
<strong>for</strong>maldehyde-saline <strong>for</strong> 24 h. <strong>The</strong>y were washed<br />
briefly in water and 10-15 µm cryostat sections were<br />
cut. Sections were mounted on to a blackened<br />
photographic plate, cut to a suitable size. <strong>The</strong> plate was<br />
prepared by exposing rapid panchromatic quarter-<br />
plate to daylight <strong>for</strong> 10-15 min, developed in the usual<br />
way and hardened in a thiosulphate-metabisulphite<br />
bath be<strong>for</strong>e drying. After mounting, the plates were<br />
allowed to become dry and then the sections and the<br />
surrounding gelatin were lightly dampened with 0.15<br />
M phosphate buffer (pH 7.6). <strong>The</strong> sections were then<br />
incubated <strong>for</strong> 30-60 min at 37°C in a petri dish<br />
All the chemicals used in the present study were<br />
procured from Sigma Chemicals, USA or SRL, India.<br />
RESULTS<br />
Controls: Intense AcPase and AlkPase activities were<br />
observed in the tegument and gastrodermis. Other<br />
regions viz., the subtegument and somatic<br />
musculature displayed staining intensity lesser than
48 Anthelmintic efficacy of genistein<br />
JPD : Vol. 28 (1), 2004<br />
Table I: Activities of AcPase, Alkpase, ATPase and 5'Nu in various structures of Fasciolopsis buski in vitro: histochemical localization<br />
Treatment<br />
AcPase AlkPase ATPase 5'-Nu<br />
(mg/ml) T ST SM IN T ST SM In T ST SM IN T ST SM IN<br />
Control<br />
(0.9% PBS)<br />
++++ +++ +++ ++++ ++++ ++ +++ ++++ +++ ++ ++ ++ +++ +++ +++ +++<br />
F. vestita ++ + + ++ + - + ++ - - - - - - + -<br />
crude root peel<br />
extract (20)<br />
Genistein (0.5) + + ++ ++ + - ++ + - - + - - - + -<br />
Oxyclozanide<br />
++ + ++ + ++ - ++ + - - - - - - - -<br />
T- tegument; ST - Subtegument; SM- somatic musculature; IN - intenstine<br />
++++ Very strong activity, +++ Strong activity, ++ Medium activity, + less activity, - No activity
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of genistein<br />
49<br />
Table II: Biochemical effects of the various plant-derived test materials on F. buski<br />
a b<br />
Treatment Enzyme activity (total /specific ) %decrease*<br />
(mg/ml) AcPase AlkPase ATPase 5'-Nu AcPase AlkPase ATPase 5'-Nu<br />
Control 11.3 ±0.26/ 29.7 ± 3.34/ 509.35 ± 85.9/ 43.3 ± 2.37<br />
(0.9% PBS) 1.48 ± 0.03 3.9 ± 0.44 66.67 ± 11.25 5.67 ± 0.31<br />
Crude extract of 6.47 ± 0.26/ 16.2 ± 1.6/ 303.72 ± 44.1/ 22.4 ± 2.14/ 42.75 44.23 39.85 47.8<br />
F. vestita (20) 0.98 ± 0.04 2.47 ± 0.25 46.22 ± 6.72 3.41 ± 0.3<br />
Genistein 6.54 ± 0.26/ 17.3 ± 2.44/ 213.9 ± 18.15/ 20.6 ± 1.87/ 42 42.31 54.5 51.9<br />
(0.5) 1.47 ± 0.06 3.9 ± 0.54 48.06 ± .08 4.6 ± 0.42<br />
Oxyclozanide 6.6 ± 0.25/ 17.46 ± 2.8/ 308 ± 27.3/ 24.84 ± 2.05/ 41.6 42.21 36.3 42.8<br />
(20) 1.2 ± 0.05 3.18 ± 0.51 56 ± 5 4.52 ± 0.4<br />
Values are given as mean (± SEM) from three to five replicate assays (5' Nu, n=4; ATPase, n=3; AlkPase, n=4; AcPase, n=5)<br />
One unit of enzyme activity is defined as that amount of enzyme which consumes 1.0 µmol substrate/g wet wt tissue/h<br />
a<br />
Specific activity expressed as unit/mg protein<br />
b<br />
* P > 0.05
50 Anthelmintic efficacy of genistein<br />
JPD : Vol. 28 (1), 2004<br />
the tegument (Table I; Figs. 1, 2, 4). Not much of<br />
activity was observed in the general parenchyma or the<br />
structures related to the excretory or reproductive<br />
systems.<br />
<strong>The</strong> ATPase activity was observed mainly in the<br />
tegument of the fluke. In the subtegument, somatic<br />
musculature and intestinal caeca moderate staining<br />
was demonstrated. Among the structures that were<br />
studied tegument, subtegument, somatic musculature<br />
and gastrodermis showed an equally strong activity of<br />
the 5'-Nu enzyme. <strong>The</strong> stain intensity was much less in<br />
the muscle layer (Figs. 7, 8, 10,11).<br />
Quantitatively the control parasites showed more<br />
enzyme activities than the treated ones with respect to<br />
all the four enzymes that were tested.<br />
Treatment: <strong>The</strong> activities of AcPase, AlkPase,<br />
ATPase and 5'Nu in the tegument and gastrodermis<br />
were inhibited after treatment with the plant test<br />
3<br />
Fig. 3: AcPase activity in the cryostat transverse sections: Reduced<br />
enzyme activity in the dorsal and ventral tegument, subtegument,<br />
somatic musculature and intestinal caecum after treatment with the<br />
root tuber peel extract of F. vestita (48 x).<br />
Fig. 1: AcPase activity in the cryostat transverse sections: Control -<br />
Intense enzyme activity in the dorsal tegument (DT), subtegument<br />
(ST) and somatic musculature (SM) (120x).<br />
1<br />
Fig. 2: AcPase activity in the cryostat transverse sections: Controlstrong<br />
enzyme activity in the intestinal caecum (IN) (120x)<br />
2<br />
4<br />
Fig. 4: AlkPase activity in the cryostat transverse sections: Intense<br />
enzyme activity in the dorsal tegument, subtegument, somatic<br />
musculature and gastrodermal lining of control flukes (48x).
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of genistein<br />
51<br />
Figs. 5-6: AlkPase activity in the cryostat transverse sections<br />
Fig 5: Enzyme activity in the intestinal caecum after treatment with<br />
the active component of F. vestita, genistein (120x).<br />
5<br />
Fig. 9: 5'-Nu activity in the cryostat transverse sections of F. buski,<br />
Mild enzyme activity detectable in sections after treatment with root<br />
tuber peel extract of F. vestita (48x).<br />
9<br />
Figs 10-11: ATPase activity in the transverse cryostat sections.<br />
6<br />
Fig 6: Reduced enzyme activity in the ventral tegument,<br />
subtegument and somatic musculature after genistein treatment<br />
(48x).<br />
Figs. 7-8: 5'-Nu activity in the cryostat transverse sections of F.<br />
buski<br />
10<br />
Fig. 10: Control- enzyme activity demonstrated in the dorsal<br />
tegument along with somatic musculature (48x).<br />
Fig.7: Control- section showing<br />
intense enzymatic activity in the<br />
tegument, subtegument and somatic<br />
musculature (48x)<br />
7<br />
8<br />
Fig. 8: Controlsections<br />
showing<br />
enzyme activity in the<br />
intestinal caecum and<br />
(48x).<br />
11<br />
Fig. 11: Control- enzyme activity in the intestinal caecum (120x).
52 Anthelmintic efficacy of genistein<br />
JPD : Vol. 28 (1), 2004<br />
Fig.12: ATPase activity in the transverse cryostat section:<br />
Diminished stain intensity in the dorsal tegument, somatic<br />
musculature and the intestinal caecum after genistein treatment<br />
(48x).<br />
materials, but moderate activity was discernible in the<br />
somatic musculature. Almost all the structures of the<br />
treated flukes showed decrease in enzyme staining<br />
intensity and a similar result was observed after<br />
treatment with the reference drug (Figs. 3, 5, 6, 9, 12).<br />
<strong>The</strong> results of the enzyme assays are described in Table<br />
II. <strong>The</strong> AcPase activity in F. buski paralyzed with the F.<br />
Fig. 13a- 13d: Protease enzyme activity in F. buski, fresh frozen<br />
sections (photographs)<br />
a<br />
a: Control b: Control<br />
c<br />
c: Treatment with F. vestita<br />
crude root peel extract<br />
12<br />
b<br />
d<br />
d: Treatment with genistein<br />
vestita crude extract and genistein was inhibited by<br />
42.75 % and 42 %, respectively, whereas oxyclozanide<br />
caused a decline by 41.6 % compared to the controls.<br />
Varying degree of inhibition of the AlkPase activity<br />
(37.5-44.23 %) in comparison to control was observed<br />
after treatment with the plant-derived components.<br />
<strong>The</strong> ATPase activity also got reduced by 39.85% and<br />
54.5% in treatments with crude peel extract of F.<br />
vestita and genistein, respectively, while in the<br />
reference drug-treated parasite a decrease by 36.6 %<br />
was recorded. Biochemical analysis of 5'-Nu showed a<br />
decrease in the enzyme activity by 47.8 % <strong>for</strong> F. vestita<br />
and 51.9 % <strong>for</strong> genistein. <strong>The</strong> parasites treated with the<br />
reference drug showed a decrease by 42.8 % (Table II).<br />
Protease: <strong>The</strong> proteolytic enzyme activity was<br />
demonstrable histochemically in both control as well<br />
as the parasites treated with the various test materials,<br />
though owing to the thickness of the sections, cellular<br />
or subcellular localization of the protease activity was<br />
not discernible. At the generalized tissue level, no<br />
conspicuous change in the intensity of the enzyme<br />
reaction was noticeable in the treated parasite from that<br />
of the control (Fig. 13 a-d).<br />
DISCUSSION<br />
Ultrastructural studies have revealed that the tegument<br />
in trematodes has an absorptive potential. <strong>The</strong><br />
processes of secretion and absorption appear to<br />
operate concurrently in the gastrodermis of most<br />
trematodes, although one or other function may<br />
predominate at any given time. In a number of<br />
helminth parasites AcPase, AlkPase, ATPase and 5'-Nu<br />
have been detected both histochemically and<br />
biochemically, and found to be closely associated with<br />
the tegument, subtegument, somatic musculature, gut<br />
and cuticle (Pappas, 1988; Leon et al., 1989; Kwak and<br />
Kim, 1996; Pal and Tandon, 1998; Fetterer and<br />
Rhoads, 2000). As revealed in the present study,<br />
alterations occurred in the enzyme activity of the<br />
parasite after treatment with the crude root tuber peel<br />
extract of F. vestita and its active component genistein,<br />
both showing synchronous effectiveness.<br />
Oxyclozanide showed more or less similar effect as the
JPD : Vol. 28 (1), 2004<br />
Anthelmintic efficacy of genistein<br />
53<br />
plant-derived components. After treatment, AcPase since studies on several epithelial cell types of<br />
showed less stain intensity compared to the controls. mammals indicate that the transport systems and sites<br />
Similar observations were made in various trematode of phosphatase activity are separate (Lumsden, 1975).<br />
species exposed to treatment with hexachlorophene Non-specific AlkPase activity was localised at the<br />
(Gupta and Sharma, 1973). Likewise, thiabendazole tegumental plasma membrane of Cyathocotyle, and in<br />
and mebendazole changed the activity of AcPase in N. the surface channels of the tegument of Schistosoma<br />
brasiliensis (Mishra and Srivastava, 1990). As in (Ernst, 1976). <strong>The</strong>se findings (of AcPase and AlkPase<br />
mammals, in helminths also, AcPase is usually in tegument and gastrodermis) raise the possibility that<br />
associated with lysosomes and AlkPase is regarded as the tegument in some trematodes may function in a<br />
indicative of membrane transport mechanisms digestive-absorptive capacity, analogous to that of the<br />
(Barrett, 1981). <strong>The</strong> inhibition of AcPase activity by mucosal lining of the vertebrate intestine. Delabrethe<br />
plant-derived components observed during the Defayolle et al. (1989) reported inhibition of AlkPase<br />
present investigation is suggestive of the fact that activity by 23% and complete inhibition of glucose<br />
absorption and intracellular digestion of drugs may uptake in Echinococcus multilocularis metacestodes<br />
involve lysosomes (Colam, 1971). <strong>The</strong> lessening of following treatment with isatin, a known phosphatase<br />
enzyme activity in the present study may probably be inhibitor, in vivo and attributed the depletion in the<br />
due to its leakage into the medium as a result of the enzyme activity to the failure of glucose uptake<br />
disruption of the absorptive surface (Hart et al., 1977). (Pappas, 1983). Some plant extracts also showed a<br />
<strong>The</strong> major AcPase activity in trematodes is localised at<br />
decrease in both AcPase and AlkPase activities in<br />
the luminal surface of the intestinal caeca and is<br />
Paramphistomum cervi in vitro (Chopra et al., 1991),<br />
associated with the plasma membrane of the lamellar<br />
but Fallon et al. (1994) showed more than two-fold<br />
folds or the microvilli (Threadgold and Brennen,<br />
increase in AlkPase activity in the PZQ-treated S.<br />
1978). It is possible that the activity at these sites is<br />
mansoni and attributed this increase to drug-induced<br />
related to digestion of phosphate esters at membrane<br />
tegumental damage exposing the normally concealed<br />
level and in common with the phosphatases of the<br />
enzymes on the tegumental surface of the worm.<br />
mammalian intestinal brush border yields a kinetic <strong>The</strong> present investigation revealed a high ATPase<br />
advantage in the subsequent absorption of the activity in the control worm. A tegumental<br />
digestive products. involvement in ionic and water regulation in F.<br />
In F. buski though histochemically AcPase showed<br />
hepatica was indicated by the demonstration at the<br />
+ +<br />
higher stain intensity compared to AlkPase,<br />
tegumental plasma membranes of Na /K -dependent<br />
biochemical results showed an increased AlkPase<br />
ATPase activity (Threadgold and Brennen, 1978). <strong>The</strong><br />
activity in the tissues compared to AcPase. In the<br />
localization of ATPase in the somatic musculature of<br />
present study AlkPase activity decreased by 37.5 to<br />
F. buski indicates that the enzyme occurs in the<br />
44.23% after treatment with the various test materials,<br />
myofibrils. <strong>The</strong> ATPase activity was inhibited by 36.6-<br />
and in histochemical localization also it showed<br />
54.5% in the flukes treated with all the test materials.<br />
decreased stain intensity. AlkPase has been localized<br />
Similar observations were also made <strong>for</strong> E.<br />
on the surface of both male and female Schistosoma<br />
multilocularis isolated scolices of hydatids after<br />
mansoni as a result of praziquantel-induced<br />
exposure to mebendazole, thiabendazole, levamisole<br />
tegumental damage (Mehlhorn et al., 1981; Modha et<br />
and acrisaline (Benediktov, 1980). Drugs like<br />
al., 1990). Any direct involvement of phosphatases in<br />
phenathiazines, chlorimazine, diethylcarbamazine<br />
absorption, as suggested by their association with<br />
and centperazine caused significant inhibition of<br />
absorptive structures in trematodes, seems unlikely<br />
ATPase of the filarial parasite, Setaria cervi,
54 Anthelmintic efficacy of genistein<br />
JPD : Vol. 28 (1), 2004<br />
indicating thereby that this enzyme system could be a Aggarwal R, Sanyal SN and Khera S. 1992. Effect of anthelmintics<br />
common target <strong>for</strong> the action of anthelmintic drugs<br />
on phosphatases in Ascaridia galli. Acta Veterinaria Hungarica.<br />
(Agarwal et al., 1990).<br />
40: 243-249.<br />
Barrett J. 1981. Biochemistry of Parasitic Helminths. Macmillan<br />
In the present study the 5'-Nu activity decreased by Publishers Ltd, London, Basingstoke.<br />
about 42-52% following treatment with genistein or Benediktov II. 1980. <strong>The</strong> effects of anthelmintics on ATPase and<br />
other test materials in F. buski. 5'-Nu is supposed to dehydrogenase system' in scoleces of Echinococcus<br />
have an active role in transport across plasma multilocularis. Naukova Dumka. 69.<br />
membrane or may be involved with other enzymes in Buchmann K. 1998. Histochemical characteristics of<br />
the hydrolysis of nucleosides to pyrimidine and purine Gyrodactylus derjavini parasitizing the fins of rainbow trout<br />
(Onchorhynchus mykiss). Folia Parasitologica (Praha). 45 :<br />
bases in parasitic nematodes (Barrett, 1981; Walter<br />
312-318.<br />
and Albeiz, 1985).<br />
Bunitian HC. 1970. Deamination of nucleotides and the role of<br />
In the present investigation protease activity was their deamino <strong>for</strong>ms in ammonia <strong>for</strong>mation from amino acids.<br />
demonstrated histochemically in F. buski, both control In: Handbook of Neurochemistry, Lajtha, A (ed.). Plenum, New<br />
and treated and there was no discernible change in the<br />
York.<br />
enzyme activity in the parasite exposed to the various Chopra AK, Sharma MK and Upadhyay VP. 1991. Effects of<br />
ayurvedic anthelmintics on phosphatase activity of<br />
treatments. <strong>The</strong> protease activity in Schistosoma is<br />
Paramphistomum cervi. <strong>Indian</strong> Journal of Helminthology. 43:<br />
reduced by specific trypsin inhibitors (Zussman and 65- 69.<br />
Bauman, 1971). As demonstrated with histochemical<br />
Colam JB. 1971. Studies on gut ultrastructure and digestive<br />
and immunocytochemical techniques a major source physiology in Cyathostoma lari (Nematoda: Strongylida).<br />
of proteolytic activity is the intestinal caeca and <strong>Parasitology</strong>. 62: 273-283.<br />
protonephridia in trematodes (Howell, 1973; Dalton JP, Neill SO, Stack, C, Collins P, Walshe A, Sekiya M. et<br />
Rotmans, 1977; Skelly and Shoemaker, 2001).<br />
al. 2003. Fasciola hepatica cathepsin L-like proteases: biology,<br />
function, and potential in the development of first generation<br />
<strong>The</strong> alterations in the activity of the tegumental liver fluke vaccines. International Journal <strong>for</strong> <strong>Parasitology</strong>. 33:<br />
gastrodermal enzymes after treatment with the various 1173- 1181.<br />
plant-derived components suggest that these enzymes Delabre-Defayolle I, Sarciron ME, Audin P, Gabrion C, Duriez T,<br />
(excluding protease) may be a potential target of action Paris J and Petavy AE. 1989. Echinococcus multilocularis<br />
<strong>for</strong> genistein, which, besides acting transtegumentally,<br />
metacestodes: Biochemical and ultrastructural investigations<br />
may as well affect through the gastrodermal interface.<br />
on the effect ofisatin (2-3 indolincdionc) in vivo. journal of<br />
Antimicrobial Chemistry. 23: 237-245.<br />
ACKNOWLEDGEMENTS<br />
Ernst SC. 1976. Biochemical and cytochemical studies of alkaline<br />
phosphatase activity in Schistosoma mansoni. In: C.P. Read<br />
This investigation was supported by a grant from Govt.<br />
memorial Rice University Studies, vol. 62: pp 81-95.<br />
of India under the DRS-III programme to the Fallen PG, Smith P, Nicholls T, Modha J and Doenhaff MJ. 1994.<br />
Department of Zoology, North Eastern Hill<br />
Praziquantel-induced exposure of Schistosoma mansoni<br />
University, Shillong.<br />
alkaline phosphatase:drug-antibody synergy which acts<br />
preferentially against female worms. Parasite Immunology. 16:<br />
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Journal of Parasitic Diseases<br />
Vol. 28 (1) June 2004, pp. 57-60<br />
Short Communication<br />
Prevalence of phthirapteran ectoparasitic insects on<br />
domestic hens of Rampur (U.P.)<br />
A. K. SAXENA*, SANDEEP KUMAR, NIDHI GUPTA AND S.K. SINGH<br />
Department of Zoology, Government Raza Postgraduate College, Rampur (U.P.) 244 901, India<br />
As many as 60.9% of the fowls (510) examined in twelve localities of district Rampur (during <strong>July</strong> 2000 to<br />
August 2002) were found infested with one or other kind of Phthiraptera. <strong>The</strong> order of prevalence of seven<br />
species has been found to be Menopon gallinae (51.3%) > Goniocotes gallinae (25.4%) > Lipeurus lawrensis<br />
tropicalis (15.8%) > Lipeurus caponis (11.5%) > Menacanthus cornutus (8.1%) > Goniodes dissimilis (7.9%) ><br />
Lipeurus heterographus (6.9%). Significant positive correlation existed between mean monthly prevalence<br />
rate and mean monthly temperature as well as photoperiod. <strong>The</strong> prevalence rate was significantly higher on<br />
birds having poor health and poor plumage.<br />
Keywords: Ectoparasite, Mallophaga, Phthiraptera, Poultry lice.<br />
he prevalence and infestation intensity of<br />
Tdifferent phthirapteran ectoparasites on certain<br />
avian hosts viz. sparrows (Hoyle, 1938; Woodman and<br />
Dicke, 1954), starlings (Boyd, 1951), blackbirds<br />
(Baum, 1968), auks (Eveleigh and Threlfall, 1976),<br />
procellarii<strong>for</strong>m birds (Fowler et al., 1984), wood<br />
ducks (Thul, 1985), reed bunting (Fowler and<br />
Williams, 1985), Wilson's petrels and storm petrels<br />
(Fowler and Price, 1987), leach's petrels (Fowler and<br />
Hodson, 1988), manx shearwaters (Fowler and Shaw,<br />
1989), five shorebird species (Hunter and Colwell,<br />
1994), house martins (Clark et al., 1994), swifts (Lee<br />
and Clayton, 1995), flycatchers (Potti and Merino,<br />
1995), peacocks (Stewart et al., 1996), spanis raptors<br />
(Perez et al., 1996), bee eaters (Kristofik et al., 1996;<br />
Hoi et al., 1998; Darolova et al., 2001) have been<br />
noted by selected workers. Only a few workers have<br />
examined birds belonging to different orders from this<br />
point of view (Ash, 1960; Klockenhoff et al., 1973).<br />
Rekasi et al., 1997 and Rozsa, 1997 have discussed the<br />
pattern in abundance of avian lice.<br />
<strong>The</strong> prevalence rate of phthirapteran ectoparasites on<br />
* Corresponding Author<br />
<strong>Indian</strong> birds is not well documented. (Chandra et al.,<br />
1990; Singh, 1999). Trivedi et al. (1992) recorded the<br />
prevalence and intensity of Phthiraptera on poultry<br />
birds of Dehradun. Prevalence of poultry shaft louse,<br />
Menopon gallinae on poultry birds of Garhwal was<br />
noted by Saxena et al., (1995). Present studies deal<br />
with the prevalence and intensity of infestation of<br />
seven phthirapteran species (Menacanthus cornutus,<br />
Menopon gallinae, Lipeurus caponis, L. lawrensis<br />
tropicalis, L. heterographus, Goniocotes gallinae and<br />
Goniodes dissimilis) on the poultry birds of Rampur<br />
district during <strong>July</strong> 2000 to August 2002.<br />
Several methods have been described <strong>for</strong> delousing the<br />
infested birds (Clayton and Drown, 2001) but during<br />
the present studies, the prevalence and infestation rate<br />
was recorded by timed visual counts as the birds<br />
belonged to the private owners / poultry keepers. <strong>The</strong><br />
surveyor had to record the prevalence and intensity<br />
without causing any harm to the bird. Hence, the bird's<br />
legs were tied with rubber band / thread and both the<br />
wings were held by the assistant (in upright position).<br />
Bird's feathers were deflected manually and the<br />
presence of lice was observed with the help of<br />
magnifying torch. Lice were taken out and placed in<br />
vials containing 70% alcohol. Each infested bird was
58 Phthirapteran ectoparasitic insects on domestic hens of Rampur (U.P.) JPD : Vol. 28 (1), 2004<br />
again subjected to 5 minutes observation (<strong>for</strong> each of birds carrying three species infestation was 16.9%.<br />
species) to record the number of lice by coding system Only a small percentage (2.4%) carried four species<br />
(1-25 lice VL, 26-50 lice L, 51-75 lice- M, 76 -100 lice infestation. Infestation by M. gallinae and G. gallinae<br />
H, and >100 lice - VH). was the most favoured combination on birds carrying<br />
two species. Likewise, the popular combination of<br />
Out of 12 species reported to occur, only seven could<br />
birds having three species was M. gallinae, G.<br />
be recovered from the poultry birds of Rampur. <strong>The</strong><br />
gallinae, and L. lawrensis tropicalis. Infestation by<br />
chicken body louse, M. stramineus (which is otherwise<br />
more than four species was not observed on the poultry<br />
very common poultry lice) was not detected on the<br />
birds of Rampur.<br />
poultry birds of Rampur. Another louse species (G.<br />
gigas) reported from poultry birds of Dehradun An attempt has also been made to find the degree of<br />
(Trivedi et al., 1992) was also not recorded. Out of 510 correlation between mean monthly lice prevalence of<br />
poultry birds examined from 12 different localities the period <strong>July</strong> 2000 to June 2001 and the mean<br />
during <strong>July</strong> 2000 to August 2002, 60.9% were found monthly temperature, photoperiod and RH of the<br />
infested with one or the other kind of poultry louse. corresponding period. Mean monthly prevalence was<br />
Species-wise prevalence and infestation intensity has 76.5% in <strong>July</strong>. It decreased to the level of 65.3% in<br />
been shown in Table.<br />
August and remained nearly constant in succeeding<br />
four months (60.0, 55.6, 58.8 and 56.5% from<br />
Analysis of the data indicates that 54.4% of male birds<br />
September to December, respectively). Prevalence<br />
carried infestation of one or the other phthirapteran<br />
showed a sharp decrease in January (48.4%) and<br />
species. Prevalence on female bird was 63.6%. Sex<br />
reached the lowest level in February (47.0%).<br />
related difference in the prevalence was not significant<br />
<strong>The</strong>reafter, it rose gradually in succeeding four months<br />
at 0 .05 level of significance. <strong>The</strong> colour of host birds<br />
till it reached the highest level in June 2001 (53.3, 75.0,<br />
was also recorded. <strong>The</strong> prevalence rate on the four<br />
80.0 and 90.0% from March to June, respectively).<br />
types of birds (white, black, brown and mixed colour)<br />
Significant positive correlation existed between mean<br />
remained 36.6%, 60.8%, 72.3% and 73.1%,<br />
monthly prevalence and mean, monthly temperature<br />
respectively. <strong>The</strong> colour related differences were not<br />
as well as photoperiod (r = 0.79 and 0.76 respectively).<br />
found significant at 0.05 levels.<br />
However, correlation with mean monthly relative<br />
Out of the 60.9% infested birds, 17.9% carried single humidity (r = -0.50) was not found significant, at 0.05<br />
species infestation. Maximum percentage of bird level of significance.<br />
(23.9%) showed two species infestation. Prevalence<br />
Table : Intensity of infestation of seven phthirapteran species on poultry of Rampur, during <strong>July</strong> 2000<br />
to August 2002.<br />
Species Prevalence Intensity of infestation<br />
VL L M H VH<br />
M. gallinae 51.3 5.5 13.9 8.4 9.6 13.9<br />
G. gallinae 25.4 3.3 10.8 3.3 4.5 3.5<br />
L. l. tropicalis 15.8 1.4 6.5 1.8 3.7 2.4<br />
L. caponis 11.5 1.6 5.7 1.2 1.4 1.6<br />
M. cornutus 8.1 2.4 4.3 0.8 0.4 0.2<br />
G. dissimilis 7.9 2.2 5.7 - - -<br />
L. heterographus 6.9 0.2 6.5 0.2 - -
JPD : Vol. 28 (1), 2004<br />
Phthirapteran ectoparasitic insects on domestic hens of Rampur (U.P.)<br />
59<br />
<strong>The</strong> degree of harmfulness of different species of difference in prevalence were not significant. Most of<br />
poultry lice (in terms of loss of weight, vitality and the workers did not find any correlation with feather<br />
productivity of host birds) is quite variable. M. colour of host bird and the louse prevalence / intensity.<br />
stramineus was not found upon poultry birds of the Studies relating to prevalence rate of lice (M. gallinae)<br />
district Rampur, but prevalence of the other dreaded on domestic fowls of Garhwal (Saxena et al., 1995)<br />
poultry louse (M. gallinae) was highest on the birds of also indicate similar results.<br />
this area. Nearly, half of the examined hosts carried<br />
Present studies further indicate that most of the heavily<br />
this louse species (51.3%). Prevalence of this louse on<br />
and very heavily infested birds encountered during the<br />
poultry birds of Dehradun and Garhwal were noted as<br />
present investigation carried M. gallinae, G .gallinae<br />
44.7% and 69.3% respectively (Trivedi et al., 1992;<br />
and also L. lawrensis tropicalis. Birds heavily infested<br />
Saxena et al., 1995). However, the prevalence of<br />
with M. cornutus and L. caponis were rarely found.<br />
another haematophagous poultry louse, M. cornutus<br />
On the other hand, any bird carrying heavy or very<br />
(whose degree of harmfulness and disease<br />
heavy infestation of G. dissimilis and L. heterographus<br />
transmission capabilities have yet not been<br />
were not observed during the survey work.<br />
investigated) remained comparatively lower (8.1%).<br />
<strong>The</strong> order of prevalence of phthirapteran species ACKNOWLEDGEMENTS<br />
recorded by Trivedi et al. (1992) was<br />
M. gallinae—44.7% > M. cornutus—40.4% ><br />
<strong>The</strong> authors are thankful to the Principal, Govt. Raza<br />
M. stramineus—26.2% > G. gallinae —19.2% ><br />
P.G. College, Rampur <strong>for</strong> providing laboratory<br />
G. dissimilis—14.3% > L. caponis—13.8% ><br />
facilities.<br />
L. lawrensis tropicalis—9.2% and > REFERENCES<br />
G. gigas—4.8%. Thus, the prevalence of<br />
Ash JS. 1960. A study of the Mallophaga of the birds with<br />
M. cornutus was quite high on poultry birds of particular reference to their ecology. Ibis. 102: 93-110.<br />
Dehradun. <strong>The</strong> prevalence of G. gallinae and L.<br />
Baum H. 1968. Biologie und okologie der Amselfederlouse.<br />
lawrensis tropicalis have been found comparatively<br />
Angew. Parasitol. 9: 129-175.<br />
higher on the poultry birds of Rampur while that of L.<br />
caponis and G. dissimilis remained comparatively<br />
Boyd EM. 1951. A survey of parasitism of the starling, Sturnus<br />
vulgaris L. in North America. J. Parasitol. 37: 56-84.<br />
lower. Lastly, Trivedi et al. (1992) did not find L.<br />
heterographus on the poultry birds of Dehradun.<br />
Chandra S, Agarwal GP and Saxena AK. 1990. Seasonal changes in<br />
a population on Menacanthus eurysternus (Mallophaga :<br />
Selected workers have noted the impact of host sex on Amblycera) on the common Myna, Acridotheres tristis. Int. J.<br />
prevalence of avian lice. <strong>The</strong>re are conflicting reports<br />
Parasitol. 20 : 1063-1065.<br />
on the subject. It has been stated that lice are more Clark F, Farrell J and Hill LA. 1994. A study of a population of the<br />
prevalent on male birds in case of sparrows (Woodman House martin (Delichon urbica (L.)) feather louse Brueelia<br />
gracilis Nitzsch (Mallophaga : Ischnocera) in Lincolnshire,<br />
and Dicke, 1954), black birds (Baum, 1968) and 45<br />
U.K. Entomologist 113: 198-206.<br />
species of variety of birds (Ash, 1960). However, no<br />
Clayton DH and Drown DM. 2001. Critical evaluation of five<br />
difference in louse prevalence with respect to host sex<br />
methods <strong>for</strong> quantifying chewing lice (Insecta : Phthiraptera).<br />
has been noted in case of alcids (Eveleigh and J. Parasitol. 87: 1291-1300.<br />
Threlfall, 1976), domestic hens (in case of M.<br />
Darolova A, Hoi H, Kristofik J and Hoi C. 2001. Horizontal and<br />
gallinae)(Saxena et al., 1995) and European beevertical<br />
ectoparasite transmission of three species of<br />
eaters (Hoi et al., 1998). During the present studies, Mallophaga and individual variation in European bee eaters<br />
sex related differences in the prevalence rate were not (Merops apisater). J. Parasitol. 87: 256-262.<br />
found to be significant. Thus, it may be stated that Eveleigh ES and Threlfall W. 1976. Population dynamics of lice<br />
some host factors may occasionally cause variation in (Mallophaga) on auks (alcidae) from Newfoundland. Can. J.<br />
louse prevalence in some cases, but generally there is Zool. 65: 2998-3005.<br />
no significant difference in prevalence with respect to Fowler JA and Hodson D. 1988. <strong>The</strong> Mallophaga of Leach's Petrels<br />
host sex.<br />
Oceanodroma leucorhoa from North Rona, Scotland. Seabird<br />
11: 47-49.<br />
Present studies further indicate that host colour related
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Fowler JA, Miller CJ and Cohen S. 1984. Non haematophagous Ecol. Ent. 20: 43-50.<br />
ectoparasites populations of Procellarii<strong>for</strong>m birds in Shetland,<br />
Perez JM, Martinez IR and Cooper JE. 1996. Occurrence of<br />
Scotland. Seabird 7: 23-30.<br />
chewing lice on spanis raptors. Ardeola 43: 129-138.<br />
Fowler JA and Price RA. 1987. A comparative study of the<br />
Potti J and Merino S. 1995. Louse loads of pied flycatchers: effects<br />
Ischnoceran Mallophaga of the wilson's petrel Oceanites<br />
of host sex, age condition and relatedness. J. Avian Biol. 26:<br />
oceanicus and British storm petrel Hydrobates pelagicus.<br />
203-208.<br />
Seabird 10: 43-49.<br />
Rekasi J, Rozsa L and Kiss BL. 1997. Patterns in the distribution of<br />
Fowler JA and Shaw GJ. 1989. <strong>The</strong> Mallophaga of Manx<br />
avian lice (Phthiraptera : Amblycera : Ischnocera). J. Avian<br />
shearwaters Puffinus p. puffinus from Ynys Enlli, Wales.<br />
Biol. 28: 150-156.<br />
Seabird 12: 14-19.<br />
Rozsa L. 1997. Pattern in the abundance of avian lice (Phthiraptera<br />
Fowler JA and Williams LR. 1985. Population dynamics of<br />
: Amblycera , Ischnocera). J. Avian Biol. 28: 249-254.<br />
Mallophaga and acari on reed bunting occupying a communal<br />
winter roost. Eco. Ent. 10: 377-383.<br />
Saxena AK, Kumar A, Surman and Singh SK. 1995. Prevalence of<br />
Menopon gallinae Linne. (Phthiraptera : Amblycera) on<br />
Hoi H, Darolova A, Konig C and Kristofik J. 1998. <strong>The</strong> relation<br />
poultry birds of Garhwal. J. Parasitic Dis. 19: 69-72.<br />
between colony size, breeding density and ectoparasitic loads<br />
of adult European bee eaters (Merops apiaster). Ecoscience 5: Singh SK. 1999. Ecology of phthirapterans infesting pigeon in<br />
156-163. Dehradun. Ph. D. <strong>The</strong>sis, H.N.B. Garhwal University, Srinagar<br />
(Garhwal) pp.150.<br />
Hoyle W. 1938. Transmission of poultry parasites by birds with<br />
special reference to the “English” or house sparrow and Stewart IRK, Clark F and Petrie M. 1996. Distribution of chewing<br />
chickens. Trans. Kansas Acad. Sci. 41: 379-384.<br />
lice upon the polygynous peacock, Pavo cristatus. J. Parasitol.<br />
82: 370-372.<br />
Hunter JE and Colwell MA. 1994. Phthirapteran infestation of five<br />
shorebird species. Willson Bull. 106 : 400-403.<br />
Klockenhoff H, Rheinwals G and Wink M. 1973. Mallophagen<br />
befall bei vogeln, Massenbefall als Folge von Schaden an den<br />
Wirten. Bonn. Zool. Beitr. 24: 122-133.<br />
Kristofik J, Manas P and Sustek K. 1996. Ectoparasites of bee<br />
eaters (Merops apiaster) and arthropods in its nests. Biologia,<br />
Bratislava 51: 557-570.<br />
Lee PLM and Clayton D. 1995. Population biology of swift (Apus<br />
apus) ectoparasites in relation to host reproductive success.<br />
Thul JE. 1985. Parasitic arthropods of wood ducks, Aix sponsa L.,<br />
in the Atlantic flyway. J. Wildlife Dis. 21: 316-318.<br />
Trivedi MC, Saxena AK and Rawat BS. 1992. Incidence of<br />
Mallophaga on poultry in Dehradun (India). Angew. Parasitol.<br />
33: 69-78.<br />
Woodman WL and Dicke RJ. 1954. Population fluctuation of the<br />
mallophagan parasite Brueelia vulgata (Kellogg) upon the<br />
sparrow. Trans. Wisconsin Acad. Sci. 43: 133-135.
Journal of Parasitic Diseases<br />
Vol. 28 (1) June 2004, pp. 61-64<br />
Stilesia daulatabadensis N.Sp. from Capra hircus<br />
V.P. SHELKE* AND G.B. SHINDE<br />
Department of Zoology, Dr. B.A. Marathwada University, Aurangabad - 431004, MS. India<br />
<strong>The</strong> present communication deals with a new species Stilesia daulatabadensis from a goat Capra hircus. It<br />
differs from all the known species of the genus with the characters : scolex globular with large, oval suckers,<br />
mature segment squarish, broader than long, testes 11 in number (7 on poral side and 4 on aporal side) cirrus<br />
slightly curved, ovary a single mass, medium, oval, vagina long, posterior or anterior to the cirrus pouch,<br />
ootype small, round, ventrolateral to the ovary, paruterine organs, two in each mature segment, in a<br />
developing stage.<br />
Key words : Capra hircus, Cestodes, Par Uterine organ, Stilesia<br />
Short Communication<br />
he genus Stilesia was erected by Railliet in 1893, or posterior half of the segments, cirrus thin, slightly<br />
Tfrom Ovis aries in Europe, Asia and Africa, as curved vas deferens medium, thin, may or may not be<br />
Stilesia globipuntata. Later on 14 species are added to coiled.<br />
this genus by various workers in the world. <strong>The</strong> present<br />
1- Scolex<br />
communication deals with the description of a new<br />
2- Mature Segment<br />
species as Stilesia daulatabadensis n.sp<br />
1<br />
3<br />
3 - 2Gravid Segment Segment<br />
Twenty specimens of cestodes, of the genus Stilesia<br />
were collected from the intestine of a goat Capra<br />
hircus. All cestodes were preserved in 4% <strong>for</strong>malin and<br />
stained with Harris Haematoxylin. Drawings are made<br />
with the help of camera lucida. All measurements are<br />
in millimeters (mm).<br />
Stilesia daulatabadensis n.sp. (Fig. 1,2,3)<br />
Scolex medium, globular rostellum absent, suckers<br />
large oval, arranged in two pairs, occupy major portion<br />
of scolex, neck long, elongated.<br />
<strong>The</strong> mature proglottids medium, thin squarish, broader<br />
than long with blunt round projection at the anterior<br />
and posterior corners of segments, acrespedote testes<br />
small to medium, oval, 11 in number 7 on poral side<br />
and 4 on aporal side bounded laterally by longitudinal<br />
excretory canals, cirrus pouch medium, oval, anterior<br />
* Corresponding Author<br />
1<br />
2<br />
1.0 mm<br />
0.5 mm<br />
1<br />
2,3<br />
3
62 Stilesia daulatabadensis N.Sp. JPD : Vol. 28 (1), 2004<br />
<strong>The</strong> ovary is medium, oval; a single mass, at 1/3rd to globipunctata. Later on the following 14 species are<br />
1/4th from the lateral margin of the same on each side, added to this genus.<br />
vagina thin tube, long, placed posterior or anterior to 2. S. vittata Railliet, 1896<br />
the cirrus pouch, slightly wavy, reaches and opens into 3. S. hepatica Wolffhugel, 1903<br />
the ootype.<br />
4. S. okapi Leiper.1935<br />
<strong>The</strong> genital pores are medium, oval, situated in the 5. S. leiper Kadam, Shinde and Jadhav, 1980<br />
anterior half of the segments, the paruterine organs are 6. S. caballeroi Kalyankar,Deshmukh &<br />
two in number, in each mature segment, in a<br />
Hatwalkar,1981<br />
developing stage, on each side, dumb-bell shaped, 7. S. southwelli Shinde, Jadhav and Kadam, 1982<br />
placed laterally in central medulla.<br />
<strong>The</strong> gravid segments are medium, squarish, almost 4 8. S.aurangabadensis M.A. Majid, G.B. Shinde, and B.V.<br />
times broader than long with convex lateral margin,<br />
Jadhav, l982<br />
with two paruterine organs in each segment. 9. S. garhwalensis Malhotra S.K; and Capoor V.N. 1983<br />
Table 10.S.kotdwarensis Malhotra S.K. and Capoor V.N. 1983<br />
Measurements of S. daulatabadensis<br />
11. S marathwadaensis Shinde, Jadhav and Phad, 1985<br />
Parts Measurements (mm)<br />
12.S. alli S.N. Borde and G.B. Shinde, 1999<br />
Length Breadth 13. Stilesia yavalensis G.B. Shinde and A.T. Kalse 1999<br />
Scolex 0.738 - 0.818 0.670 - 1.011 14. S. dhondagae S.B. Deshmukh and L.V. Shinde, 2001<br />
Suckers 0.2,05 - 0.375 0.363 - 0.375 After going through the literature the worm under<br />
Neck 0.767 0.112-0.214 discussion differs as follows :<br />
Mature Segment 0.131 - 0.189 1.195 - 1.199<br />
<strong>The</strong> worm differs from S. caballeroi in the latter<br />
Testes 0.029 - 0.049 0.019 - 0.039 having 1-11 testes on each side, vas deferens<br />
Cirrus Pouch 0.112 0.029 - 0.058 anterior to the testes, less dense bundle of<br />
Cirrus 0.112 0.005 convolutions, cirrus pouch oval, vagina posterior to<br />
Vas Deferens 0.194 0.005 - 0.010 cirrus pouch, paruterine organ spherical on<br />
Ovary 0.087 - 0.092 0.072 - 0.097 postolateral surface and from the host Capra<br />
Vagina 0.262 0.005<br />
hircus.<br />
Genital Pore 0.019 0.024 - 0.034 <strong>The</strong> present worm differs from S. kotdwarensis in<br />
Para Uterine Organs 0.180 - 0.223 0.092-0.112 the latter having 1-12 in number, ovary small<br />
Gravid Segment 0.150 - 0.184 0.762 - 0.781<br />
spherical, paruterine organ small, 2 in number,<br />
between dorsal and ventral logitudinal excretory<br />
vessels from the host Ovis aries.<br />
Type species Stilesia daulatabadensis n.sp.<br />
Host<br />
Capra hircus<br />
It differs from S. alli in the latter having scolex<br />
round or oval, mature segments small or squarish,<br />
Habitat Intestine<br />
testes medium round 11 in number ( i.e. 5+6 = 11,<br />
Locality At Daulatabad,<br />
6+5 =11 ) cirrus pouch medium situated in the<br />
Tq. Khultabad, Dist. Aurangabad, M.S., India<br />
anterior half of the segment, ovary large oval single<br />
Date of collection 25th Febuary, 1996 mass, paruterine organ bag like oval two in each<br />
<strong>The</strong> genus Stilesia was erected by Railliet in 1893, gravid segment, found in Capra hircus.<br />
from Ovis aries in Europe, Asia and Africa, a Stilesia
JPD : Vol. 28 (1), 2004<br />
Stilesia daulatabadensis N.Sp.<br />
63<br />
It differs from S. dhondagae in the latter having testes<br />
8-10 in number, arranged in the two groups, cirrus<br />
pouch small, oval, situated middle to the posterior side<br />
of the segment, ovary distinctly bilobed, elongated<br />
having 8-9 acini and from the host Capra hircus.<br />
Hence the above noted characters are valid enough to<br />
erect a new species <strong>for</strong> these worm and the name<br />
Stilesia thapari n.sp. is proposed in honour of Dr. G.S.<br />
Thapari, Ex - Professor and Head, Department of<br />
Zoology, Lucknow University, who has contributed a<br />
lot in our knowledge of Helminthology<br />
Majid MA, Shinde GB and Jadhav BV. 1982. On a new species<br />
Stilesia Railliet 1893 (cestoda :Thysanosomatinae<br />
skrjabin,1933) from sheep at Aurangabad Marath. Univ. J. Sci.<br />
(Nat. Sci.) XXI 14 : 37 - 39.<br />
Malhotra SK and Capoor VN. 1983. On two new species of<br />
cestodes (cyclophyllidea), Stilesia garhwalensis n.sp. from<br />
goat and Stilesia kotdwarensis n.sp. from sheep of the Garhwal<br />
region. India. Acta. Parasit. Pol. 28 : 399 - 406.<br />
Raillet 1896 - Surquelques Parasites des clromadise. Compt. Rend.<br />
Soc. Biol. 51 : 18 - 21.<br />
Shinde GB, Kadam SS and Jadhav BV. 1982. On a new cestode<br />
Stilesia southwelli n.sp. from goat at Aurangabad, India.<br />
Marath. Univ J. Sci.<br />
ACKNOWLEDGEMENTS<br />
Shinde GB, Jadhav BV and Phad AN. 1985. Stilesia<br />
<strong>The</strong> Authors are thankful to the Professor and Head of<br />
marathwadensis n.sp. (Cestoda:Thysanosomatinae Skrjabin,1<br />
933) from Capra hircus at Aurangabad. Riv Parasit 2(46) : 213-<br />
Zoology Department. Dr. B.A.Marathwada 215.<br />
University, Aurangabad, <strong>for</strong> providing necessary Shinde GB and Kalse AT. 1999. On a new tapeworm Stilesia<br />
laboratory facilities.<br />
yavalensis (Cestoda; thysanosomidae, Fuhrmann, 1907)<br />
REFERENCES<br />
Sp.Nov from Capra \hircus in India. Uttar Pradesh J. Zool., 19<br />
(1):89-91, 1999<br />
Bhalerao GD. 1 934. On the occurance of Stilesia vittata (cestoda) Wolffhugel K. 1903. Stilesia hepatica nov.sp.ein Bandwum aus<br />
in ovines in India. Ind. J. Vet. Sci. and Anim. Husbn. 5(1 ) : 28- den Gallengangen von Schafen and Ziegen Ostafrikas, Berlin<br />
29. Tiearazi. Wochenschr. 43:661 =665.<br />
Borde SN, Shinde GB. 1999. One new species Stilesia alli n.sp.<br />
(Cestode : Thysanosominae, Fuhmiann,1907) from Capra<br />
hircus at Hasnabad Dist. Jalna, India. Uttar Pradesh J. Zool.,<br />
19(3):189-191.<br />
Deshmukh S.B. and Shinde L.V. 2001 - New tapeworm from Capra<br />
hircus at Kaij dist. Beed (M.S.) India. Rivista di parassitologia,<br />
vol. XVIII (LXII) : 171 - 175<br />
Gough. 1911. A monograph of the tapeworms of subfamily.<br />
Avitellinae, being a review of the genus Stilesia and an account<br />
of the histology of Avitelline centri-punctata. Riv. Quart. J.<br />
Micro. Sci. 56 : 317 - 383.<br />
Kadam SS, Shinde GB, Jadhav BV. 1980. On a new species of<br />
Stilesia railliet,1893 cestoda Thysanosomatinae)<br />
skrjabin,1953, from sheep at Aurangabad. Biol - vol II No. 3 :<br />
33 - 36.<br />
Kalyankar SD, Deshmukh AL and Hatwalkar VM. 1981. A new<br />
species of the genus Stilesia Riailliet, 1893<br />
(Anoplocephaloidea : Cestoda) from goat, Capra hircus at<br />
Aurangabad Biol. 3 (1 ): 51 - 52<br />
Kumar S and Lal SS. 1986. Effect of implantation of Stilesia<br />
globipuncNata (cestoda : Anoplocephalidae) scolex on the<br />
host, intestine Ind. J. Parasit (1986), 10-20 ;161 -163.<br />
Leiper 1935 - Report on the helminth parasites of the Okapi living<br />
in the <strong>Society</strong>'s Gardens Proc. Zool. Soc. London 1935, 11-12.<br />
Yamaguti S. 1959. Systema Helminthum Vol. II. <strong>The</strong> Cestode of<br />
Vertebrates. Interscience Publisher, New Yorkand London,<br />
p:339.
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accept the article, return it <strong>for</strong> revision, decline it or<br />
offer to review it after revision. All articles submitted<br />
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paper requiring revision is not returned within 3<br />
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Authors are responsible <strong>for</strong> the accuracy of their<br />
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INSTRUCTIONS TO AUTHORS<br />
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Acknowledgements : Acknowledgement should be<br />
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unpublished work should be incorporated in the text<br />
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than two authors and semi colon between references<br />
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<strong>The</strong> following style should be followed:<br />
Reference - Journal<br />
In the list of references at the end of the paper, all the<br />
names of authors should be given. However if there are<br />
more than 6 authors, list first 6 names followed by the<br />
phrase "et al".<br />
Trager W and Jensen JB. 1976. Human malaria<br />
parasite in continuous culture. Science. 193 : 673-675.<br />
Cheever AW, Macedonia JG, Mosimann JE and<br />
Cheever EA. 1994. Kinetics of egg production and egg<br />
excreted by Schistosoma mansoni and S. japonicum in<br />
mice infected with a single pair of worms. American<br />
Journal of Tropical Medicine and Hygiene. 50 : 281-<br />
285.<br />
<strong>The</strong> name of the journal should either be given full or<br />
abbreviated as per the index medicus or medline.<br />
Reference - Books<br />
Schaniz PM and Kagan IG 1980. Echinococcosis. In :<br />
Immunological Investigations of Tropical Parasitic<br />
Diseases. Houba.V (Edt.) Churchill Living stone, New<br />
York. pp 104-129.
Reference-Conferences<br />
Mahajan RC, Malla N, Stella M and Ganguly NK<br />
1995. Cysticercosis in India: Immunodiagnosis and<br />
Treatment. Paper presented at the Twelfth National<br />
Congress of <strong>Parasitology</strong>. Panaji, Goa, India, 23-25<br />
January.<br />
Reference-reports<br />
World Health Organization, 1984. <strong>The</strong> Leishmaniasis.<br />
Technical Report Series No. 701. Geneva,<br />
Switzerland.<br />
Tables: Tables should be typed separately and<br />
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Commissions should be used. At the first citation in the<br />
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Letters to the Editor : This is the quickest way of<br />
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SUBMISSION OF MANUSCRIPTS<br />
Three copies of the manuscript together with duly<br />
filled "Undertaking by Authors" along with a brief<br />
covering letter should be sent to:<br />
Prof. Nancy Malla<br />
Editor In Chief, Journal of Parasitic Diseases,<br />
Head Department of <strong>Parasitology</strong>,<br />
PGIMER,<br />
Chandigarh-160 012, India
JOURNAL OF PARASITIC DISEASES<br />
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Undertaking by Authors<br />
We, the undersigned give an undertaking to the following effect with regard to our research article entitled:<br />
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submitted <strong>for</strong> publication in the Journal of Parasitic Diseases<br />
1. Research reported was conducted within the proper animal care and according to applicable National Laws.<br />
2. <strong>The</strong> article mentioned above has not been submitted <strong>for</strong> publication in any <strong>for</strong>m to any other Journal.<br />
3. <strong>The</strong> paper if published in the Journal of Parasitic Diseases will become the sole copyright of the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong><br />
<strong>Parasitology</strong> and the article(s) thereof will not be published elsewhere unless written consent is obtained from the Editor.<br />
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Please note:<br />
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2. No addition/deletion/or any change in the sequence of the authorship will be permissible at a later stage, without valid<br />
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3. If the authorship is contested at any stage, the article will be either returned or will not be processed <strong>for</strong> publication.<br />
Important<br />
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1. Manuscripts are prepared as per the <strong>for</strong>mat of JPD.<br />
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<strong>The</strong> <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong><br />
1973<br />
Membership Application<br />
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Membership Fee<br />
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 />
Membership of the <strong>Society</strong> includes subscription to the Journal of Parasitic Diseases, a biannual publication<br />
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Membership is governed by the Constitution and Bye Laws of the <strong>Indian</strong> <strong>Society</strong> <strong>for</strong> <strong>Parasitology</strong>.<br />
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Note: Duly completed <strong>for</strong>m along with draft should be mailed to Dr. J.K. Saxena Secretary, ISP, Division of<br />
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Personal cheques/cash are not accepted. <strong>The</strong>re is no annual or ordinary membership. This <strong>for</strong>m may be xeroxed.
Forthcoming Scientific Events<br />
Date/Location : September 2-4, 2004; Warsaw<br />
Title : XX Congress of the Polish Parasitological <strong>Society</strong><br />
In<strong>for</strong>mation : Dr. Anna Rocka, Department of Medical Biology, Medical University of Warsaw,<br />
Nowogrodzka Street 73, 02-018 Warszawa, Poland.<br />
E-mail/Ph./Fax : lchomicz@ib.amwaw.edu. pl., Phone/Fax: (48-22)-625-2468<br />
Date/Location : September 23-26,2004; Mumbai, India<br />
Title : 50 Years of Medical Writing - International Conference on Journal Writing<br />
and Publishing<br />
in<strong>for</strong>mation : Dr AtuI Goel. Department of Neurosurgery, Seth G. S. Medical College. Parel.<br />
Mumbai -400 012. INDIA<br />
E-mail/Ph./Fax : goldcon@jpgmonline.com.Phone: 91-22-24129884. Fax: 91-22-25032398<br />
Date/Location : September 26 - October 8, 2004; Montevideo, Uruguay.<br />
Title : Course on Pathogen Trypanosomes - Mammalian Host-cell<br />
Interactions: Biochemistry, Cell Biology And Prospects For Drug Development<br />
In<strong>for</strong>mation : Center <strong>for</strong> Free Radical and Biomedical Research (CFRBR). Departamento de<br />
Bioqulmica. Facultad de Medicina. Universidad de la Republica, Montevideo. Uruguay.<br />
Website : www.hhmi.org/grants/awards/inst/intl_courses.html<br />
Date/Location : September 27-29,2004; Boston<br />
Title : XXXVI Annual <strong>Society</strong> <strong>for</strong> Vector Ecology (SOVE) Conference<br />
In<strong>for</strong>mation : Major Dhillon<br />
E-mail/Ph./Fax : Phone: 909/340-9792<br />
Website : www.sove.org.<br />
Date/Location : September 28-29,2004; Boston<br />
Title : American <strong>Society</strong> of Tropical Medicine and Hygiene (ASTMH), Intensive<br />
Update Course in "Clinical Tropical Medicine and Travelers' Health"<br />
E-mail/Ph./Fax : astmh@astmh.org:; Phone: 847/480-9592; Fax: 847-480-9282<br />
Website : www.astmn.org.<br />
Date/Location : September 30 - October 3, 2004; Boston<br />
Title : XLII Infectious Diseases <strong>Society</strong> of America (IDSA) Annual Meeting<br />
In<strong>for</strong>mation : IDSA, 66 Canal Center Plaza (Suite 600), Alexandria, VA22314<br />
E-mail/Ph./Fax : Fax: 703/299-0204<br />
Website : www.idsociety.org
Date/Location : October 7 -8, 2004<br />
Title : Vaccines 3: Frontiers in vaccine development Institut Pasteur<br />
In<strong>for</strong>mation : www.pasteur.fr/applications/euroconf/vaccines3/index.html<br />
E-mail/Ph./Fax : euroconf@pasteur.fr ; Fax: 33()1 40 61 3025<br />
Date/Location : October 30 - November 2. 2004; Washington. DC<br />
Title : IVL Inter science Conference on Antimicrobial Agents and<br />
Chemotherapy (ICAAC)<br />
In<strong>for</strong>mation : ASM Managed Meetings, 1752 N Street, NW, Washington. DC 20036-2904.<br />
E-mail/Ph./Fax : conferences@asmusa.org: Phone: 202/942-9261: Fax: 202/942-9340;<br />
Website : www.asm. org/meetings/index<br />
Date/Location : November 7 - 11, 2004; Miami<br />
Title : Llll American <strong>Society</strong> of Tropical Medicine and Hygiene (ASTMH)<br />
Annual Meeting<br />
In<strong>for</strong>mation : ASTMH Secretariat. 60 Revere Drive (Suite 500), Northbrook, IL 60062<br />
E-mail/Ph./Fax : astmh@astmh.org; Phone: 847/480-9592; Fax: 847/480-9282<br />
Website : www.astmh.org<br />
Date/Location : November 12 - 14, 2004; Galveston, Texas<br />
Title : McLaughlin Symposium - <strong>The</strong> Changing Landscape of Vaccine Development:<br />
Translating Vaccines <strong>for</strong> Emerging Diseases and Biodefense to the Marketplace<br />
Website : www.utmb.edu/scvd. ( Note: For additional in<strong>for</strong>mation, see <strong>PDF</strong> file.)<br />
Date/Location : November 14 - 20, 2004; Southampton, Bermuda<br />
Title : American <strong>Society</strong> of Microbiology (ASM) Conference on "DNA Repair<br />
and Mutagenesis"<br />
In<strong>for</strong>mation : ASM Managed Meetings. 1752 N Street. NW, Washington, DC 20036-2904<br />
E-mail/Ph./Fax : conferences@asmusa.org; Phone: 202/942-9261; Fax: 202/942-9340<br />
Website : www.asm.org/ meetings/index.<br />
Date/Location : Nov 17-20, 2004<br />
Title : 5th Louis Pasteur Conference on Infectious Diseases Institute Pasteur<br />
E-mail/Ph./Fax : clp@pasteur.fr<br />
Website : www.pasteur.fr/infosci/conf/sb/CLP5/<br />
Date/Location : <strong>July</strong> 10-13, 2005; Singapore<br />
Title : V International Conference on "Urban Pests (ICUP)<br />
In<strong>for</strong>mation : ICUP 2005 Secretariat, 73 Bukit Timah Road, Rex House (Suite 03-01),<br />
Singapore 229832<br />
E-mail/Ph./Fax : info@icup2005.com.sg; Phone: (65)-6330-6830; Fax: (65)-6336-2123;<br />
Website : www.icup2005.com.sg.
Date/Location : September ??-??, 2005; Marseilles<br />
Title : XVI International Congress <strong>for</strong> "Tropical Medicine and Malaria and<br />
Centenary of Tropical Medicine Institute <strong>for</strong> the French Army"<br />
Website : www.iftm.org.<br />
Date/Location : December 11 - 15, 2005; Washington, DC<br />
Title : LIV American <strong>Society</strong> of Tropical Medicine and Hygiene (ASTMH)<br />
Annual Meeting<br />
In<strong>for</strong>mation : ASTMH Secretariat, 60 Revere Drive(Suite 500), Northbrook, IL 60062<br />
E-mail/Ph./Fax : astmh@astmh.org; Phone: 847/480-9592; Fax: 847/480-9282<br />
Website : www.astmh.org.<br />
CONGRATULATIONS!<br />
Prof. Md. Hafeez, Deptt. of <strong>Parasitology</strong>, College of Veterinary<br />
Science, ANG Ranga Agriculture University, Tirupati has been awarded<br />
the distinction of "Environmentalist of year award-2003" by the<br />
'International Board of Awards of "National Environmental Science<br />
Academy", New Delhi. <strong>The</strong> award was presented to him at XVII Annual<br />
Conference of the Academy and International Workshop on 'Recent<br />
Trends in Environmental Sciences and Eco Journalism', held in New<br />
Delhi from 24 -26 April, 2004.
Third National Workshop on<br />
"Simple Diagnostic Methods in Infectious Diseases"<br />
7th -11th December 2004<br />
Department of Microbiology<br />
Jawaharlal Institute of Postgraduate Medical Education & Research,<br />
Pondicherry<br />
<strong>The</strong> number of Participants will be restricted to 20<br />
<strong>The</strong> Last date of receipt of applications is 30th September 2004<br />
For details please contact<br />
Course Coordinator:<br />
Dr. Subhash Chandra Parija MD, PhD<br />
Professor & Head<br />
Department of Microbiology, JIPMER<br />
Pondicherry - 605006<br />
Telephone: Off: 0413-2272380-90, Extn.:3200,<br />
Residence: 0413-2253016; Mobile: 0413 3114418<br />
Telegram: JIPMER, Fax: 0413-2272067<br />
E-mail: idworkshop@rediffmail.com<br />
<strong>The</strong> Selected Participants have to submit a registration fee of Rs. 1,500/- (Rupees One<br />
thousand and five hundred only) <strong>for</strong> the workshop and may be sent in as a bank draft payable to<br />
"Course-Coordinator, Infectious Disease Workshop" at Pondicherry.<br />
Note: <strong>The</strong> cost of boarding and lodging is not included in the registration fee. Accommodation<br />
will be provided in JIPMER guesthouse on a double room-sharing basis. <strong>The</strong> selected<br />
participants have to pay an additional Rs. 1,000/-(Rupees One thousand Only) towards<br />
accommodation and boarding expenditure and is mandatory.<br />
For further details about the workshop log on to the web site www.jipmer.edu
JOURNAL OF PARASITIC DISEASES<br />
Volume 28 Number 1 June 2004<br />
CONTENTS<br />
PRESIDENTIAL ADDRESS<br />
<strong>The</strong> Perfect Match - Parasite & host: Made <strong>for</strong> each other 1<br />
M.S. Jairajpuri<br />
REVIEW<br />
Role of DNA microarray Technology <strong>for</strong> understanding differential 5<br />
gene expression in Parasitic Diseases<br />
A.Debnath, A. Sen, James H. Mckerrow and P. Das<br />
EPIDEMIOLOGY<br />
Epidemiological studies on bovine microfilariasis in coastal districts 17<br />
of Andhra Pradesh<br />
V. Pavan Kumar, B. Sreedevi, T. Venkata Reddy<br />
and K. Nalini Kumari<br />
Seasonal occurrence of helminth parasites in Schizothorax in 23<br />
Dal Lake Kashmir<br />
A.R. Khan, M.Z. Chishti, Fayaz Ahmad, Majidah Rashid and<br />
Shafqat Bakshi<br />
DIAGNOSIS<br />
A monoclonal antibody to 120 kDa B. malayi antigen with 29<br />
diagnostic potential in Bancroftian filariasis<br />
Balaji Ganesh, B. Parab P.B., Katdare M., Reddy M.V.R.<br />
and Harinath B.C.<br />
CHEMOTHERAPY<br />
Anthelmintic efficacy of extract of Stephania glabra and aerial root 37<br />
extract of Trichosanthes multiloba in vitro: two indigenous plants in<br />
Shillong, India.<br />
V. Tandon, L.M. Lyndem, P.K. Kar, P.Pal, B. Das and H.S.P. Rao<br />
Anthelmintic efficacy of genistein, the active principle of 45<br />
Flemingia vestita (Fabaceae): Alterations in the activity of<br />
the enzymes associated with the tegumental and gastrodemal<br />
interfaces of the trematode, Fasciolopsis buski<br />
Pradip Kumar Kar and Veena Tandon<br />
SHORT COMMUNICATION<br />
Prevalence of phthirapteran ectoparasitic insects on domestic hens 57<br />
of Rampur(U.P.)<br />
A.K. Saxena, Sandeep Kumar, Nidhi Gupta and S.K. Singh<br />
Stilesia daulatabadensis N. Sp. from Capra hircus 61<br />
VP Shelke and GB Shinde<br />
- Editorial Policy & Instructions to Authors<br />
- Undertaking by Authors<br />
- Membership Application<br />
- Forthcoming Scientific Events<br />
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