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Volume 28
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June 2004
ISSN : 0971-7196
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Published By
The Indian Society for Parasitology
Electronic version available on ISP Website
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JOURNAL OF PARASITIC DISEASES
(ISSN : 0971-7196)
Patron
Prof. M. Shamim Jairajpuri,
President, The Indian Society for Parasitology
&
Professor, Department of Zoology Aligarh Muslim University, Aligarh - 202002, India
Editor-in-Chief
Prof. Nancy Malla,
Department of Parasitology
PGIMER, Chandigarh
Managing Editor
Dr. M.L. Dubey
Department of Parasitology
PGIMER, Chandigarh
Dr. A.B. Chowdhury
Kolkata
Dr. N.K. Ganguly
New Delhi
Dr. V.S. Chauhan, New Delhi
Dr. N.J. Shetty, Bangalore
Dr. B.C. Harinath, Sevagram
Dr. Durdana S. Jairajpuri, Aligarh
Dr. Q.H. Baqri, Jodhpur
Dr. D.P. Haldar, Kalyani
Dr. Md. Hafeez, Tirupati
Dr. V.M.L. Srivastava, Lucknow
Dr. S.C. Parija, Pondicherry
Advisory Board
Dr. R.C. Mahajan
Chandigarh
Editorial Board
Dr. V.P. Sharma
New Delhi
Dr. G.P. Dutta
Lucknow
Dr. Chetan Chitinis, New Delhi
Dr. R. Madhavi, Vishakhapatnam
Dr. A.R. Khan, Srinagar
Dr. Sobhna Sharma, Mumbai
Dr. Buddhadev Mana, Kolkata
Dr. Ch. Dhanachand, Manipur
Dr. Jagdish Mahanta, Dibrugarh
Dr. B. Jadhav, Aurangabad
Dr. R. Madhubala, New Delhi
Journal of Parasitic Diseases is published biannually by the Indian Society for Parasitology in June and December
in each calender year. The subscription price for libraries and other multi reader organizations for each number is Rs. 400 in
India and U.S. $ 100 elsewhere. Subscription by Demand Draft in favour of 'The Indian Society for Parasitology' should be sent
to Dr. J.K. Saxena, Secretary, The Indian Society for Parasitology, Division of Biochemistry, Central Drug Research Institute,
Chattar Manzil, Lucknow - 226 001 (India).
The Indian Society for Parasitology
Executive Committee
(2002-2004)
President
Prof. M.S. Jairajpuri, Aligarh
Vice-Presidents
Dr. J. Mahanta, Dibrugarh
Dr. V.M.L. Srivastava, Lucknow
Secretary
Dr. J.K. Saxena, Lucknow
Jt. Secretary
Dr. T. Adak, New Delhi
Treasurer
Dr. L.M. Tripathi, Lucknow
Members
Prof. Irfan Ahmad, Aligarh
Prof. Q.H. Baqri, Jodhpur
Dr. A.P. Dash, Bhubaneshwer
Dr. Neeru Singh, Jabalpur
Prof. N.J. Shethy, Bangalore
Dr. V.S. Chauhan, New Delhi
Dr. Prakash Babu, Hyderabad
Prof. Kaleysa Raj, Trivandrum
Dr. B.V. Jadhav, Aurangabad
Dr. Neelima Gupta, Bareilly
Statements and opinions expressed in the Journal of Parasitic Diseases or in the presentations during the regular
meeting of the Indian Society for Parasitology are those of the author(s) and do not necessarily reflect the official position of
the Society. The Editorial Board, Publisher and the Society disclaim any responsibility for the accuracy of statements made
by the contributors.
Copyright ©2004 The Indian Society for Parasitology

JOURNAL OF PARASITIC DISEASES
Volume 28, Number 1, June 2004
OFFICIAL ORGAN OF
THE INDIAN SOCIETY FOR PARASITOLOGY
Head Office : Central Drug Research Institute, Lucknow - 226 001, India
Editorial Office : Department of Parasitology, Post Graduate Institute of Medical Education and Research,
Chandigarh-160 012 India
Fax : 0172-2744401, E-mail : medinst@pgi.chd.nic.in

Journal of Parasitic Diseases
Copyright © 2004 The Indian Society for Parasitology
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No part of this publication may be reproduced or utilized in any form or by any means, electronic or
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JOURNAL OF PARASITIC DISEASES
Volume 28 Number 1 June 2004
CONTENTS
PRESIDENTIAL ADDRESS
The Perfect Match - Parasite & host: Made for each other 1
M.S. Jairajpuri
REVIEW
Role of DNA microarray Technology for understanding differential 5
gene expression in Parasitic Diseases
A.Debnath, A. Sen, James H. Mckerrow and P. Das
EPIDEMIOLOGY
Epidemiological studies on bovine microfilariasis in coastal districts 17
of Andhra Pradesh
V. Pavan Kumar, B. Sreedevi, T. Venkata Reddy
and K. Nalini Kumari
Seasonal occurrence of helminth parasites in Schizothorax in 23
Dal Lake Kashmir
A.R. Khan, M.Z. Chishti, Fayaz Ahmad, Majidah Rashid and
Shafqat Bakshi
DIAGNOSIS
A monoclonal antibody to 120 kDa B. malayi antigen with 29
diagnostic potential in Bancroftian filariasis
Balaji Ganesh, B. Parab P.B., Katdare M., Reddy M.V.R.
and Harinath B.C.
CHEMOTHERAPY
Anthelmintic efficacy of extract of Stephania glabra and aerial root 37
extract of Trichosanthes multiloba in vitro: two indigenous plants in
Shillong, India.
V. Tandon, L.M. Lyndem, P.K. Kar, P.Pal, B. Das and H.S.P. Rao
Anthelmintic efficacy of genistein, the active principle of 45
Flemingia vestita (Fabaceae): Alterations in the activity of
the enzymes associated with the tegumental and gastrodemal
interfaces of the trematode, Fasciolopsis buski
Pradip Kumar Kar and Veena Tandon

SHORT COMMUNICATION
Prevalence of phthirapteran ectoparasitic insects on domestic hens 57
of Rampur(U.P.)
A.K. Saxena, Sandeep Kumar, Nidhi Gupta and S.K. Singh
Stilesia daulatabadensis N. Sp. from Capra hircus 61
VP Shelke and GB Shinde
Editorial Policy & Instructions to Authors
Undertaking by Authors
Membership Application
Forthcoming Scientific Events

Journal of Parasitic Diseases
Vol. 28 (1) June, 2004, pp. 1-4
Presidential Address
3rd Global Meet on Parasitic Diseases
(January 12-16, 2004, Bangalore)
The Perfect Match
Parasite & Host : Made for each other
M. Shamim Jairajpuri
President, Indian Society for Parasitology
Department of Zoology, Aligarh Muslim University
Aligarh -202002, India
he Darwin's Theory of Evolution states that
Tevery species produces excessively large number
of offsprings much more than their own existing
populations. But the Natural Selection allows only
those individuals with suitable variations to survive.
The ecosystem, however, requires population
constancy of the entire component species. This raises
the important question as to how does the Nature
manage these two rather contrasting phenomena?
Further, what is the actual mechanism by means of
which the unwanted rise in the population level is
managed and brought down? The answer is that it is the
parasites, pathogens, parasitoids, pests and the
predators which may be called as 5 'Ps' that carry this
out and for this reason they are often regarded as
"Nature's Hangmen". These organisms thus play a
very significant role in population balance and they
have been performing this duty ever since this living
world has come into existence.
The food is basic necessity for all organisms. Various
feeding types and interactions have evolved between
different animal and plant species during the course of
evolution. These may be microphagous,
saprophagous, phytophagous, carnivorous,
predaceous, etc. In parasitism, species or group of
organisms called parasites do not have the ability to
obtain food directly from the ecosystem; consequently
they have to depend on some other organisms (host)
for food. The physiological compatibility between the
two is such that quite often the host and the parasite coevolve
though not necessarily at the same pace. The
parasite-host interactions may occur among animal-
animal-plant-plant and these provide some very
interesting associations in different types of
organisms. Consequently, there is a need to look into a
few of these aspects.
All organisms prefer to live in peace and harmony in
their ecosystems. However, for their survival and to
obtain food they may be responsible for causing death
of others, if the situation so demands. But this is a part
of Nature's overall strategy for maintaining an
ecological balance. The host-parasite associations are
unique in the sense that of the two organisms it is only
the parasite that is benefitted while the host suffers.
However, the parasite also does not gain in anyway by
the death of its host because this amounts to destroying
its own source of food. The perfect situation, to the
advantage of the parasite, would indeed be to obtain its
food without causing any serious or significant
damage to the host. Such an ideal situation is not
always known to exist but theoretically all parasitehost
associations ultimately progress in this direction.
It is precisely due to this reason that perfectly sound
host-parasite combinations of species co-evolve and
co-exist. Further, closely related species of hosts are
known to possess very similar to often identical
species of parasites. In developing harmony with the
host, the parasite species also has to synchronize and
adapt itself to a great extent not only structurally and
ecologically but physiologically and biochemically as
well. In making adjustments to suit each other, the host
and the parasite become a perfect match leading to
synchronization in their adult and larval stages of
development. But the term matching or made for each

2 Jairajpuri : Presidential Address
JPD : Vol. 28 (1), 2004
other should not lead someone to believe that the
parasitism in anyway is synonymous either with
commensalism or symbiosis. In the latter association
actually none of the organisms involved suffer any
kind of disadvantage or injury. The same is not true in
parasitism as the host species definitely suffers
varying degrees of injury, which may be insignificant
or temporary inconvenience to some sort of discomfort
or disease which may even lead to the death of the host
organism.
It is evident that the adult stages of the parasite no
doubt adapt themselves to a life in or on their host and
in due course of time gradually attain a state of
equilibrium with them. Of special importance are the
life stages (young ones/larvae/juveniles) of the
parasites and the various strategies that these may
adapt to gain access/entry into their hosts. Some of this
could be done directly but in others an intermediate
host, a vector or carrier may be involved. An extreme
kind of parasitism is exhibited by the parasitic
crustacean, Sacculina which initially has a nauplius
larva much like any other species of Crustacea but this
soon changes into a cypris larva with a bivalve shell
like that of the cirripedes. This larva attaches itself to
one of the setae of the crab host and it soon throws off
its limbs and becomes reduced to a mass of
undifferentiated cells enclosed in the skin. This results
into total loss of all organs of the parasite except the
reproductive. For feeding, Sacculina develops rootlike
processes which penetrate into various organs of
the crab body. It is evident that the parasites in order to
accommodate themselves to their host make great
adjustments. In the present case, Sacculina has
sacrificed its total identity.
An interesting and quite noteworthy correlation exists
in the life history of the monogenetic trematode,
Polystoma integrrimum with its host, the frog. The
parasite attaches itself to the transparent walls of the
urinary bladder of frogs. The fluke lays its eggs during
the spring and its onchomiracidia are ready almost at
the same time when the tadpoles of the frog are
attaining the internal gill stage of their development.
The little ciliated larva then gets attached to the
tadpole. As the gills change into lungs and the frog
moves towards adulthood, the fluke also becomes a
young adult almost simultaneously and settles down in
the bladder. This shows how beautifully the life cycles
of the parasite and its host are matched. In case the
onchomiracidia hatch early and get attached to the
external gills of the tadpole, these become adults
quickly, but outside the body of frog. Consequently,
they perish without reaching the urinary bladder. In
much the same way, the Protozoa Balantidium,
Opalina and Nyctotherus which live inside the rectum
of frog produce cysts only during spring time when the
tadpoles are also present in the pond to feed upon them.
This way the infection of the parasites passes on
successfully from one generation of the host to the
other.
The filarial worms that parasitize human beings and
animals provide one of the finest examples of
synchronization in the life cycle between the host and
its parasite. Wucheraria bancrofti, the Bancroft's
filarial worm produces its young ones called
microfilaria which appear in the peripheral blood
circulation during the night when the host is asleep.
This coincides with the timing of the visit of
mosquitoes, the intermediate host. As mosquitoes bite
and suck blood, the microfilaria pass into their bodies
and subsequently develop into the infective stages.
The latter are transferred back to man when the
mosquitoes bite again for their blood meal. However,
nocturnal periodicity would change into diurnal in
those persons who work by the night and rest/sleep
during the daytime. The same will happen, if an
infected person happens to travel halfway round the
globe. Another filarial worm called eye worm, Loa loa
has diurnal periodicity because its intermediate host
are day-biting tabanid flies, Chrysops sp. The fiery
serpent, called the guinea or the Medina worm,
Dracunculus medinensis is also a good example of a
nematode species which parasitizes man. The females
upon maturity contain millions of juveniles which are
to be liberated in water so as to reach their intermediate
host, the Cyclops. The females begin crawling towards
the hand and feet, which are most likely to come in
contact with water. Biologically, it is a very interesting
phenomenon and one wonders as to how do these
females know that their best chance of liberating the
juveniles in water is to move towards extremities of the
host. However, in those people who carry water on

JPD : Vol. 28 (1), 2004
Jairajpuri : Presidential Address
3
its body. During evolutionary process and in order to
mitigate the severity of the host-parasite struggle, both
the parasite and the host make all possible efforts to
adapt to each other. The parasite causing less and less
damage and the host gradually getting used to the
presence of the parasite. Consequently, equilibrium is
reached in this association when the parasite inflicts
minimum or no damage to the host.
From the foregoing account let no one get the
impression that I am eulogizing the parasites in
anyway. What I have desired to convey is the fact that
they are also an important component of our ecosystem
and that is the way they are going to be come what may.
Practically every animal and plant species is
parasitized by one or more species of parasites. Man
alone is parasitized by some 36 different species of
nematodes. Add to this list, the species of flukes,
tapeworms, insects and the microbial organisms for
which we the human beings are the most favored host.
Nobody would dispute that in recent times man has
caused great destruction to the world ecology. An
exponential rise in human population has also
contributed to a great extent to environmental
degradation and in the rise of pollution levels the
world-over particularly in the Third World countries.
Many of the parasitic diseases that had declined or
almost disappeared in the past such as Malaria have
come back with a vengeance. We need to evolve global
strategies to deal with parasitic diseases as the
parasites do not respect man-made national
boundaries. The first step in this direction would be the
environmental management of parasites and to make
efforts to determine the weakest points in their life
cycles which could be exploited to tackle them more
effectively. The world-wide efforts to develop vaccine
against malarial parasite are indeed laudable but we
must also direct our efforts more vigorously towards
breaking the life cycle of mosquitoes in their aquatic
phase of life. This would be far easier and cheaper and
simultaneously it may also help in combating other
mosquito-transmitted diseases. Though multi-
pronged efforts would also prove effective for certain
parasites but the cost-benefit factor should not be
ignored before embarking on such a strategy. The
Developed World has already succeeded to a great
extent in combating parasitic diseases in their
their back the females migrate to this area rather than to
the extremities.
The mermithid nematodes produce very peculiar type
of eggs that have long filamentous branches. These get
entangled to the plants so as to hold the eggs. The
grasshoppers /locusts become infected while eating
grasses and foliage containing the eggs. Similarly, the
larval cercariae of Fasciola hepatica, the liver fluke
encyst on grasses and other type of vegetation waiting
to be eaten and ingested by sheep or other definitive
hosts. The infective juveniles of Agamermis
decaudata emerge from the soil cavities and climb
nearby vegetation especially during periods of high
moisture such as dew or rain. There they wait for the
newly hatched insect nymphs and penetrate their
hemocoel through the body wall as and when they
come in their contact. One could go on like this quoting
innumerable interesting example of host-parasite
interactions with particular reference to their mutual
compatibility.
All parasitic associations initially begin only as a
matter of chance contact between two species of
organisms. None of these is a parasite to start with,
though one of them happens to be smaller and weaker
then the other one. The sole intention of the weaker
organism is to feed on the larger one, ecto- or
endoparasitically. The future generations of these two
species maintain this interaction and thus a new
parasite-host association begins. During the initial
period of interaction, the parasite will still have the
ability to survive on its own in the absence of its host
because it is only in the facultative and not the obligate
parasitic stage. But in due course of evolution and
struggle for survival, the parasite also gradually
becomes more and more dependent on the host.
Ultimately, a stage reaches when the parasite can not
survive without the host. At this point, the parasite has
already become obligatory in nature and it is now
entirely dependent upon its host. While the parasite is
gradually in the process of evolving from the
facultative to the obligatory stage, the host simply does
not remain a silent spectator. Simultaneously, it also
makes efforts using its tissue reactions and the
mechanism of resistance or immunity to fight off the
parasitic invasion thereby minimising the damage to

4 Jairajpuri : Presidential Address
JPD : Vol. 28 (1), 2004
countries. But the people in the Developing World and purposes. Further, a lot of man-hours are lost annually
more so in the Underdeveloped World are grappling due to the health problems of working population
with these health problems in a big way not only for which could otherwise have been gainfully utilized.
man himself but also for his live-stock, the agricultural This is beside the yearly deaths that are caused due to
and horticultural crops. Huge sums of money is spent parasitic diseases. It should be our endeavor to free
every year from the already depleted resources of these humanity from the ill effects of diseases due to
countries in fighting off parasitic and other diseases parasites. May this Global Meet turn out to be an
which could have been spent for development important step in this direction !

Journal of Parasitic Diseases
Vol. 28 (1) June 2004, pp. 5-16
Review
Role of DNA Microarrary Technology for understanding
differential gene expression in Parasitic Diseases
1 1
ANJAN DEBNATH , ABHIK SEN, JAMES H. MCKERROW AND PRADEEP DAS*
National Institute of Cholera & Enteric Diseases, P-33, C.I.T Road, Scheme-XM, Kolkata-700010, India
1,
and Sandler Center for Basic Research in Parasitic disease University of California,
San Francisco, CA 94143, USA
DNA microarray holds exceptional promise as a technique to exploit the explosion of genomic information.
It is, in principle and practice, an extension of hybridization-based methods that have been used to identify
and quantitate nucleic acids in biological samples. But unlike other techniques, it allows for tens of
thousands of genes to be represented in areas smaller than standard glass microscopic slides called 'chips'.
Samples of interest are fluorescently labeled and allowed to hybridize to the array; after hybridization
followed by washing steps, an image of the array is acquired and the representation of individual nucleic acid
species in the sample is reflected by the amount of hybridization to complementary DNAs immobilized in
known positions on the array. Microarrays are typically constructed based on either sequence information
obtained from genome sequencing projects or by arraying inserts from a random fragment genomic library.
The technique is now widely used in understanding the host response as well as identification of pathogen
virulence determinants. It has also been successfully employed in the study of differential gene expression in
disease and health, high-throughput detection of single-nucleotide polymorphisms, genome mapping,
mutation screening; and identification of potential vaccine targets. Among the parasites, microarray
analysis has been recently applied to study various aspects ,of the pathogenesis and life cycles of Plasmodium
falciparum, Toxoplasma gondii, Leishmania spp., Cryptosporidium parvum, Trypanosoma cruzi,
Trypanosoma brucei, Entamoeba histolytica, Schistosoma mansoni, Ascaris suum and Trichostrongylus
vitrinus. To illustrate the utility of this technique in gene expression studies some of the results of E.
histolytica and human collagen interaction will be discussed.
Keywords: Array, Collagen, Entamoeba histolytica, Genome, Parasites
P
arasitic diseases have a debilitating impact on
human health worldwide, particularly in
developing countries. Malaria, Schistosomiasis,
Leishmaniasis and Trypanosomiasis still result in a
large number of deaths each year, while drug
resistance in malaria is increasing as a public health
problem (Ellis et al 2003). Among the gut-dwelling
parasitic protozoa, Entamoeba histolytica and Giardia
intestinalis are frequent causes of acute diarrhoea
while intracellular parasite Cryptosporidium parvum
is commonly associated with diarrhoea in infants. In
agriculture, the steadfast presence of gastrointestinal
* Corresponding Author
parasites in livestock throughout the world is
responsible for significant economic loss. Control
measures against gastrointestinal nematodes alone
cost over 1 billion pound annually (McDonald, 2003)
but resistance in parasitic worms is increasing at such
an alarming rate that many drugs will soon be rendered
useless to the livestock industry.
In order to identify new genes with key cellular
functions, targets for new drugs, and new antigens
which could be useful in the diagnostic or for vaccine
development, scientists from developing and
developed countries planned and initiated a number of
parasite genome projects. Thus, the genomes of E.
histolytica, Giardia lamblia, Plasmodium falciparum,
Schistosoma mansoni, Trypanosoma cruzi,

6 DNA Microarray in parasitic diseases
JPD : Vol. 28 (1), 2004
Leishmania major, \Trypanosoma brucei, Brugia virulence determinants that enable E. histolytica to
malayi and other pathogenic nematodes, are now cause invasive colonic and hepatic disease have not
under study (Degrave et al 2001). Access to all open been identified and the genetic factors that make E.
reading frames from the genome of these organisms is histolytica virulent and E. dispar avirulent remain to
imminent, and a fully annotated genome of parasites be elucidated. With the advent of microarray technique
will soon be available. Sequence function in itself has our understanding of the complex genetic processes
already become too complex to be dissected by the underlying the interaction between Entamoeba and the
classical reductionist approach to molecular biology, host will be accelerated and consequently, will provide
where genes are analyzed individually (Rathod et al valuable insight into design of novel means to prevent,
2002). Moreover, DNA sequences alone tell us little treat and diagnose infection.
about the dynamic processes within the parasite.
Therefore, the task facing parasitologists is to extract
WHAT ARE DNA MICROARRAYS?
useful information from rapidly accumulating genome A microarray is a high-density microscopic
data (Ashton et al 2001). Functional genomics has arrangement of nucleic acid targets immobilized on a
wider application in identifying potential protein solid substrate. Samples of interest are fluorescently
targets for vaccination or novel drug targeting. Global labelled and allowed to hybridize to the array.
profiling of gene expression is one attractive approach Following hybridization an image of the array is
to assessing function. Because a gene is usually acquired and the representation of individual nucleic
transcribed only when and where its function is acid species in the sample is reflected by the amount of
required, determining the locations and conditions hybridization to complementary DNAs immobilized
under which a gene is expressed allows inferences in known positions on the array (Eisen and Brown,
about its function. Several independent high- 1999).
throughput methods, such as serial analysis of gene
expression (SAGE), differential display and expressed MICROARRAY FABRICATION
sequence tag (EST) analysis, are available for gene
Microarraying of double-stranded or single-stranded
expression studies and may enable function annotation
DNA fragments on a glass microscopic slide is being
of sequenced genomes. But DNA microarray
used extensively owing to the accessibility, cost
technology stands out for its simplicity,
effectiveness and proven success of this approach.,
comprehensiveness, data consistency, and high Four broad variations of DNA microarray are now
throughput (Cummings and Relman, 2000). The
being constructed in parasite laboratories worldwide:
purpose of this review is to focus on the general
methodology for microarray development with special Random (shotgun) microarrays
emphasis on a recently developed E. histolytica
These have been constructed either out of a mungmicroarray
highlighting the applications of
bean-nuclease digested genomic DNA (gDNA) library
microarray in understanding the molecular basis of
or from a randomly sheared (using nebulizer) gDNA
host-pathogen interaction of different parasites.
library. In most slides, duplicate spots are attached as
Amoebiasis, caused by the protozoan parasite E. controls, either next to or distant from the first spot,
histolytica continues to be the major cause of with up to 20,000 spots per slide. In gene-expression
morbidity and mortality. WHO in its most recent experiments, mRNA samples are converted to cDNA
estimates has placed the death toll from amoebiasis at and are fluorescently labelled with flurochrome dyes,
40,000-100,000 lives annually (WHO, 1997). Cy5 (red) and Cy3 (green), either during reverse
Significant progress has been made in understanding transcription or afterwards. The two samples are then
the mechanisms that make these intestinal protozoa incubated with the microarray slide and the
unique in its ability to cause invasive disease in fluorescently labelled probe DNA hybridizes to the
humans. Also, the genetic mysteries of the organisms immobilized target DNA with high sensitivity and
are beginning to be revealed; helping to explain the specificity. The slide is scanned and the intensity of red
pathogenic difference versus closely related and green fluorescence at each spot is proportional to
commensal organisms such as Entamoeba dispar the amount of Cy5 and Cy3-labelled cDNA
(Stauffer and Ravdin, 2003). But still the majority of respectively that has hybridized to the DNA at that

JPD : Vol. 28 (1), 2004
DNA Microarray in parasitic diseases
7
spot. This quantitative ratio between the two DEVELOPMENT OF E. histolytica SHOTGUN
fluorescent dyes gives information about the MICROARRAY
comparative over expression or under expression of
genes in an experimental sample. The advantage of
As the genome of E. histolytica remains to be fully
competitive hybridization is that the ratio remains the
sequenced, it is currently not possible to construct
same even if there is differences in the amount of target
gene-specific microarrays representing complete E.
DNA present (Bryant et al 2004). Since the
histolytica genome. However, given the relatively
sequences of the arrayed gDNA fragments on a
small size of the E. histolytica genome, valuable
shotgun microarray are unknown, it necessitates
information on gene expression related to
post-hybridization sequencing of interesting
environmental signals can be initially assessed by
clones. Shotgun gDNA microarrays offer the
microarray analyses. The E. histolytica genome has
possibility of an immediate, genome-scale short intergenic regions and relatively few and short
experiment even in the absence of a complete
introns. This makes the use of genomic library for
genome sequence.
microarray development a feasible approach (Singh
and Shah, 2002). Random shotgun collections provide
Gene-specific microarrays
an efficient means for generating DNAs capable of
identifying large fraction of Entamoeba mRNAs, and
These are being developed from large sets of wellare
therefore well suited for expression profiling
characterized gene sequence tag (GST) and EST
studies. It is relatively inexpensive to prepare DNA
libraries. Such arrays have the advantage of starting
collections, and expression-profiling analysis need
with known sequences. The representation of genomic
not wait for the availability of complete genomic DNA
sequences could be biased in EST sequences because
sequence (Akopyants et al 2001). Due to the random
the library is usually derived from a specific stage of
nature of the shotgun cloning procedure, there is an
parasite life cycle. Nonetheless, an enormous amount
unbiased representation of sequences in the genomic
of fresh information can be derived from such arrays
library, so genes that are rarely transcribed have the
(Rathod et al 2002).
same representation as highly expressed ones. Overall
Double-stranded ordered arrays
genome representation is usually good in shotgun
libraries with comparatively little variation across the
Following publication of full-length sequences of genome.Thus, even if randomly selected clone inserts
different chromosomes, a physical map of annotated are used as probes, there should be good coverage and
genes in a specific linear order is now available. relatively little redundancy. Also, not only coding but
Amplified genes from genome template using also intergenic regions can be studied (Diehl et al 2002).
sequence-specific primers are being arrayed alongside
other gDNA fragments to provide additional To begin to identify global changes in E. histolytica
insights into transcriptional regulation mechanisms gene expression related to different stimuli, we
(Rathod et al 2002),
produced an E. histolytica microarray using a
variation of the standard array technology.
Long oligonucleotide array
Approximately 1 kb inserts from an E. histolytica
Long oligonucleotide can be spotted directly on slides.
random fragment genomic library were isolated to
Over the entire slide there are several different
generate 'shotgun' microarrays (Debnath et al 2004).
oligonucleotide sequences from the same gene. This Array construction
range produces more robust results because the same
gene can be probed independently several times in the Genomic DNA was sheared using the nebulizer
same experiment. This approach is simpler because it (Invitrogen, USA) connected to the laboratory
bypasses the laborious PCR, DNA purification and gel compressed air line. The sheared DNA was blunt-end
analysis steps of traditional array construction, and the repaired with T4 DNA and Klenow polymerases, and
exact sequence of the 70mer DNA strand are known for the ends were dephosphorylated with calf intestinal
®
each spot (Rathod et al 2002). It also allows the user to phosphatase to create a suitable substrate for TOPO
avoid genes that are repetitive or homologous to other cloning (Invitrogen). DNA fragments were ligated into
® ®
known genes.
pCR 4Blunt-TOPO vector (Invitrogen) and the

8 DNA Microarray in parasitic diseases
JPD : Vol. 28 (1), 2004
cloning reaction was electroporated into One Shot®
TOP 10 Electrocomp E. coli (Invitrogen) and the
transformants were incubated overnight on agar plates
in the presence of X-gal and 100µg/ml of ampicillin
(Debnath et al 2004).
washing purpose (Holloway et al 2003c). Five
microliter PCR products from each 96 well plate are
transferred to 384-well printing plates (Genetix, USA)
using a BioMek FX 96 well pipetting robot (Beckman
Coulter, USA) and dried down in speed-vac (Savant,
USA).
Perhaps the most laborious aspect of generating
spotted DNA microarrays is the production of In order to resuspend the array-ready material before
concentrated solutions of array-ready DNA fragments printing, the optimal solvent for the DNA depends on
by high-throughput PCR. The goals are to amplify the geometry of printing tip, the printing substrate
inserts from a large percentage of clones, to avoid being used, the length of time the plates are exposed
cross-contamination, and to purify the fragments from during printing, and the degree and type of
the enzymes, nucleotides, primers, and other environmental control available in the printing device.
components of PCR. To maximize the yield from a Moreover, the volume of solution in the wells of the
single round of PCR amplifications and to enable the printing plates is important because overly full wells
printing of thousands of microarray slides, PCRs are can lead to excessive wetting of the exterior surface of
carried out in a volume of 100µl for each gene or pins and poor spot morphology as well as dripping.
microan-ay element. The. yield of PCR is significantly Considering all these factors, 4 (µl of 3X SSC was
improved by use of fresh cultures as a source of found to be the best solvent for DNA printing and also
template DNA (Holloway etal 2003a).
the required minimum volume to obtain consistent
filling of the printing pins. DNA concentrations in the
DNA yield, the presence of free primers or primer printing solution are typically in the range of 100-250
dimers, and the number of bands in each reaction are
µg/ml because there is a loss of sensitivity and an
important measures of the quality of PCR-amplified
material available for printing. Quality control is
performed prior to or after purification of PCR
products. Analysis before purification avoids the cost
and effort involved in processing PCRs (Holloway et
al 2003b). Commercially available gel systems (E-
Gel® 96 gel, Invitrogen) provide rapid efficient
analysis. PCR products are assayed via 96 well agarose
gel electrophoresis. Although in some cases PCR
products may contain an extra band, a single
prominent PCR product is visible and the extra band
does not interfere with array hybridization.
underestimation of the degree of differential
expression between samples if the DNA concentration
is lower than 100µg/ml. Also, excessively
concentrated solutions waste valuable material and
can lead to smearing or comet tails in hybridization
(Holloway et al 2003d).
The purification of PCR products before the printing
of the microarray serves to remove enzymes or other
proteins that can clog printing tips, oligonucleotide
primers that can compete with the product for substrate
binding, and salts that could affect binding to the
substrate. To clean up PCR products and prepare the
DNA for printing, the PCR products are isopropanol-
precipitated. During precipitation isopropanol is used
instead of ethaaol to reduce the volume of the
precipitation mixture to an amount that will fit in the
tissue culture plates. Since some batches of absolute
ethanol (100%) had been reported to contain
fluorescent contaminants that generate high
backgrounds in microarray experiments, so 70%
ethanol is prepared from 95% ethanol for DNA
Although arrays of DNAs on porous membranes have
been in use for years to perform parallel hybridization
studies, technical advances made it possible to
transform these rather clumsy membranes into much
more useful and efficient methods to generate arrays
with high densities of DNAs, allowing for tens of
thousands of genes to be represented in areas smaller
than standard glass microscopic slides. Glass slides
have been a favoured solid support for immobilization
ofDNAs because of their easy availability, low
intrinsic fluorescence, high transparency, good
thermal properties, excellent rigidity, and
straightforward chemistries for modification of the
surface. Due to the nonporous nature of glass, the
labelled cDNAs have direct access to immobilized
DNAs without limitations of internal diffusion
(Holloway et al 2002).
The best choice for coating glass microscopic slides is
poly-L-lysine. Many alternate coating materials had
been tried, including numerous silanes, but none ating

JPD : Vol. 28 (1), 2004
DNA Microarray in parasitic diseases
9
proved to be as simple and reliable as polylysine. It is first few printed spots, giving rise to large-irregular
not only inexpensive, but also readily accessible and spots (Childs et al 2003). DNA samples in 3X SSC in
generally provides good results. The coating attaches 384-well microtiter plates are placed in the plate
firmly to the slide and binds tightly to DNA spotted holder. The robot positions a cluster of specialized
onto the surface. The binding of DNA to poly-L-lysine spring-loaded 16 or 32 printing tips into adjacent wells
is complex but essentially involves charge interactions of the DNA source plates, to a depth of about 1 mm,
(Childs et al 2003). Although most slides come in filling the reservoir slot of each tip with approximately
boxes labelled "precleaned", it is essential that they 1µ
l of DNA solution. The tips are then lightly tapped at
should be stripped clean prior to coating with alkaline identical positions on each slide, leaving a small (

10 DNA Microarray in parasitic diseases
JPD : Vol. 28 (1), 2004
between print runs, the printing materials are a free carboxylate group on the other end of the chain. In
completely dried in a vacuum concentrator at the end addition to converting the amino groups of the lysines to
of each run for storing. At the end of the print run, the amides, this new moiety creates a negatively charged
slides are allowed to dry to facilitate binding of the surface that further reduces nonspecific binding of
printed DNA and the slide coating.
DNA. As the blocking reagent has limited stability in
solution, it is important to carry out the process very
The E. histolytica array was constructed by printing
quickly. The last step during microarray post-
9600 PCR-amplified inserts from twenty five 384-well
processing consists of boiling the arrays. The boiling
plates using the arraying robot with a similarly sized
step is meant to denature the double stranded DNA
print-tip cluster of 32 tips spaced for 384-well plates.
molecules and thus enhances hybridization
The size of the genome of E. histolytica is predicted to
availability(Eisen and Brown, 1999,
be 20 Mb. The average size of the insert applied to the
http://www.microarrays.org/pdfs/PostProcessing2001
array was 1 kb and the microarray was constructed by
.pdf).
printing 9600 PCR-amplified inserts from E. histolytica
genomic DNA library. Theoretically, the microarray Use of E. histolytica microarray to identify genomic
covered 9.6 Mb or almost half of E. histolytica genome. responses by Entamoeba to collagen binding
Since the E. histolytica genome has large number of
repetitive sequences, some inserts will have identical or
Since collagen is a major component of the epithelial
nearly identical sequences. Based on these calculations,
basal lamina, and the extracellular matrix of the
we estimate that this microarray may cover one half to
intestine, E. histolytica encounters collagen during
one-third of the E. histolytica genome (Debnath et al
colonic invasion. In vitro, incubation of trophozoites
2004).
with collagen type I leads to parasite activation which
includes the release of electron-dense granules
After a complete set of samples is spotted, slides were (EDGs), containing factors considered important in
processed for hybridization, checked once again, to the pathogenicity of this parasite (Martinez-Palomo et
ensure that spotted DNA attaches to the slide stably. al 1987; Munoz et al 1991, 1996; Serrano et al 1996).
Active groups on the slide surface are deactivated, Interaction of trophozoites with human collagen type I
unbound DNA is removed and the target DNA is triggers biochemical cascades (Perez et al 1996), but
denatured for hybridization. Three steps are required to the mechanism of activation and the complement of
prepare printed arrays for hybridization: rehydration downstream products released has not been
and snap drying, blocking, and denaturation. Since the elucidated. To identify cellular mechanisms of
DNA solution dried to the outer edge of the spot during trophozoite activation, we assessed global variations
the printing process, the goal of rehydration is to allow in gene - expression during collagen interaction with
the DNA a chance to more evenly distribute across the E. histolytica. The arrays were probed with
surface of the spot. This helps to eliminate the donut- differentially labelled cDNAs prepared from total
shaped appearance of the spots and increases the RNA isolated from collagen-activated and nonamount
of total DNA bound after processing. The activated cells (Debnath et al 2004).
rehydration is a delicate process, as insufficient
rehydration can produce irregular spot sizes and can Typically, fluorescently labelled cDNA is generated
reduce overall hybridization intensity, while excessive
by incorporation of dye-conjugated nucleotide
rehydration can cause spots to fuse. Arrays on poly-Lthese
fluorescent nucleotide analogs are quite
analogs during the reverse transcription process. But
lysine coated slides require blocking of exposed amines
prior to hybridization to prevent binding of labelled
expensive and they have low efficiency of
material indiscriminately and nonspecifically to the
incorporation by polymerase. Linkage of a primary
surface. The blocking process, which is the most critical
amine to a N-hydroxysuccinimidyl ester group
step in post processing, is a competition between excess
attached to a dye (Cy3 and Cy5) provides an
DNA molecules leaving spots to bind near-by exposed
alternative probe labelling method that increases the
lysine, and the succinic anhydride used for blocking
fluorescent intensity of the final product and decreases
reacts and caps the amines. The charged amino group of
the cost. The reactive amine derivative ofdUTP, 5-(3-
the lysine carries out a nucleophilic attack on one of the
aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-
carboxyl carbons, forming an amide bond and exposing
dUTP), is used during labelling because it can be

JPD : Vol. 28 (1), 2004
DNA Microarray in parasitic diseases
11
incorporated by a variety of RNA-dependent and assign coordinates to each of the spots. The laser
DNA-dependent DNA polymerases. After removing scanner produces light with a wavelength appropriate
free nucleotides, the amino-allyl labelled samples can for the excitation spectra of the dyes used (for Cy3 this
be coupled to dye, purified again, and then applied to is green light around 540 nm; for Cy5 red light around
the microarray. For the labelling of a complex mixture 650 nm) (Eisen and Brown, 1999). To extract
of samples, as is the case with mRNA, the labelling meaningful data and also to draw logical and
density can be roughly controlled by limiting the reproducible conclusions from the massive amount of
amount of aa-dUTP in relation to the amount of dTTP. information distilled from the complex E. histolytica
This choice depends on the overall A/T content of the DNA chip data, effective tools for data mining are
genome in question. A ratio of 3 aa-dUTPs to 2 dTTPs critical. The data analysis was performed using
worked good for E. histolytica genomic DNA arrays. powerful analytical GenePix Pro 4.0.1.9 software.
Compared to enzymatic incorporation of dye, the Local background was subtracted from the value of
procedure for post-coupling of reactive dyes is a each spot on the array. Only spots in which almost
longer process both in terms.of time and steps. more than 70% of the pixels had a signal above
However, the added advantage of brightness, lack of background of at least twice the standard deviation
enzymatic bias, and low cost is likely to be worth the (S.D.) of the local background were considered in
extra effort (DeRisi, 2003). cDNA from non-activated subsequent data analysis. The data were normalized
E. histolytica was coupled with Cy3 dye and cDNA using the NOMAD microarray database.
from collagen-activated E. histolytica was coupled Normalization of expression data is performed to
with Cy5 dye.
adjust the individual hybridization intensities and to
balance them appropriately so that meaningful
During hybridization, buffer of high ionic strength is
biological comparisons can be made. It minimizes
used to reduce electrostatic repulsion and to promote
unequal quantities of starting RNA, differences in
association of complementary strands. Detergent is
labelling or detection efficiencies between the
used to minimize background noise. The hybridization
fluorescent dyes used, and systematic biases in the
mixture is heated to reduce the background (Ideker el
measured expression levels (Quackenbush, 2002).
al 2003). The probe is carefully pipetted onto the
The Cy5/Cy3 fluorescence ratios and logiocenter
of the array and a coverslip is placed in such a
transformed ratios were calculated from the
way so that formation of bubbles could be avoided
normalized values. In order to gain speciflcity and the
since air bubbles and inhomogeneities in the flatness
threshold expression level of gene that can be
of the slide and/or coverslip could cause locally
considered significant, the gene expression profile for
decreased signal. To provide humidity in the
each individual gene is established (Dhiman et al
hybridization chamber and also to ensure that the
2001). The interesting genes are visually identified
probe mixture does not dehydrate during
using a scatterplot of the data. In general, a factor of
hybridization, a larger volume of 3X SSC is added on a
two change in expression is considered to be adequate
separate part of the slide and the chambers are well
to decide whether a particular gene is upregulated
sealed. This decreases high background often
(Groen, 2001). Therefore, genes whose expression
observed near the edges of the coverslip (Eisen and
changed 2-fold or more relative to collagen-uninduced
Brown, 1999). Although optimal time for
E. histolytica were considered positive and selected
hybridization depends on the complexity and
for further sequencing experiments.
concentration of the sample, 16 hour hybridization
provided good result. Both hybridization and washing Cluster analysis is used to group together genes with
are carried out under conditions of moderate to high similar patterns of expression. Although various
stringency to suppress cross-hybridization.
clustering methods can usefully organize gene
expression measurements, the resulting ordered but
After hybridization, a fluorescent image is acquired
still massive collection of numbers remains difficult to
for both the fluorescent dyes used by a scanner
assimilate. The end product is a representation of
(Figure1). The photomultiplier tube (PMT) voltage is
complex gene expression data that, through statistical
set in such a way so that brightest pixels are just below
organization and graphical display, allows to
the level of saturation. Increased PMT setting can
assimilate and explore the data in a natural intuitive
increase both signal and noise. Gridding helps to

12 DNA Microarray in parasitic diseases
JPD : Vol. 28 (1), 2004
The faithfulness of the shotgun DNA microarray for
reporting collagen-specific gene expression was
apparent from the fact that due to the size selection
process prior to the shotgun cloning, the fragments
were almost similar in length. Also, variation in the
efficiency of priming events was minimized, since an
identical primer pair was used in all reactions. Three
separate hybridizations from three independent cDNA
preparations showed virtually identical differential
hybridization patterns and in total, 225 clones out of
9600 white colonies printed on the DNA array were
actually upregulated (= or > 2) due to collagen
activation. Clustering the genes enhanced their
similarities and made it easy to correlate those that
might have a relationship with the specific event.
Sequencing results of initial 14 clones showed good
homology with functionally important proteins. Few
genes that were shown by the DNA microarray to be
manner. To illustrate this approach, pairwise average-
linkage cluster analysis is applied to collected gene
expression data (Figure 2). This method is a form of
hierarchical clustering. The computed trees are used to
order genes in the original data table, so that genes or
groups of genes with similar expression patterns are
adjacent. The ordered table then is displayed
graphically with a representation of the tree to indicate
the relationships among genes (Eisen et al 1998).
over expressed during collagen interaction were
validated by RT-PCR, affirming the reliability of this
microarray (Debnath et al 2004).The homology
analysis of the sequences of 14 collagen activated over
expressed cDNA fragments corresponded with SH3
domain-containing protein SH3P17 (accession no.
AAC50592), intersectin 1 (accession no. 042287),
VAV (Homo sapiens) (accession no. CAA34383),
growth factor receptor-bound protein 2 (GRB2 adapter
protein) (SH2/SH3 adapter GRB2) (ASH protein)
(accession no. Q07883), unconventional myosin IB
(E. histolytica) (accession no. AAC47535);
asparaginyl-tRNA synthetase (Mus musculus)
(accession no. XP_128957); cysteine proteinase 1 and
2 precursors (accession no.s Q01957, Q01958); E.
histolytica gene for pore-forming peptide (accession
no. X70851); E. histolytica putative GTPase and
putative RNA binding protein genes, partial cds; and
ehapt2 retrotransponson (accession no.s AY141200,
AY141201); 40S ribosomal protein S24 and ribosomal
Fig. 1. E. histolytica genomic microarray of 6144 clones.
Collagen-induced mRNA expression profile was monitored by
microarray hybridization analysis. A fluorescently labelled (Cy3)
cDNA probe was prepared from total RNA isolated from normal E
histolytica trophozoites by reverse transcription and a Cy5-
labelled second probe was prepared from equal quantity of total
RNA isolated from collagen-activated E. histolytica trophozoites
by reverse transcription. In this image, hybridization of Cy3-
labelled cDNA is represented as a green signal and hybridization
of Cy5-labelled cDNA is represented as a red signal. Thus, genes
induced or repressed due to interaction of amoebae with collagen
appear as red and green spots, respectively. Genes expressed at
roughly equal levels appear as yellow spots.
Fig. 2. A representative gene expression map from a portion of
whole microarray hybridization. Descriptions of each experiment
appear above each column (EhCSlpl-056, EhCSlp2-055 and
EhCSlp2-060), whereas gene identifiers appear to the right of each
row. Red cells indicate increased expression due to collagen
compared to non-activated trophozoites; green cells indicate
decreased expression; grey cells represent negative or undetectable
expression. The intensity of the colour corresponds to the
magnitude of change with brighter colours showing larger
differences versus "non-activated trophozoites.

JPD : Vol. 28 (1), 2004
DNA Microarray in parasitic diseases
13
protein S19 (accession no. NP_187143); E. histolytica more sophisticated Plasmodium microarray spotted
putative mitochondrial type and endoplasmic with open-reading frame-specific oligonucleotides
reticulum-like HSP70 gene (accession no.s showed a unique coordinated expression of genes
AY141198,AY141199).
encoding ribosomal proteins, multiple factors for
transcription' and translation, enzymes of biosynthetic
The probable functions of collagen-induced genes
and catabolic pathways, or merozoite adherence and
include coding for components of a signalling cascade
invasion machinery (Bozdech et al 2003).
previously hypothesized to transmit responses
induced by cell attachment, adapter proteins for The application of microarray analysis to Toxoplasma
vesicle formation, proteins implicated in cytoskeletal gondii gene expression has advanced to the analysis of
reorganization and locomotion, and tissue degradation coordinate regulation of cluster of genes. T gondii
- cysteine proteinases and the amoebapore (Debnath et cDNA microarrays were used to study changes in
al 2004).
transcript levels during tachyzoite-to-bradyzoite
differentiation (Cleary et al 2002). Cluster analysis
The molecular consequences of collagen adherence on
identified coordinate bradyzoite expression of clones
E. histolytica provide confidence that many of the
that encode cell-surface proteins and proteincollagen-responsive
genes encode proteins that are
modifying enzymes. The groups of genes that were
relevant to the virulence of the parasite. The findings
repressed in bradyzoites included several that encode
also provide an insight into collagen-induced
secretory organelle proteins and regulatory enzymes.
signalling in amoebae, and strengthen the notion that a
The same microarray, used to compare global gene
genetic program is stimulated by interaction of E.
expression in wild-type cell lines with chemically
histolytica with extracellular matrix.
induced, tachyzoite-to-bradyzoite differentiationdefective
Taken together, the development of E. histolytica
cell lines allowed the construction of a model
microarray helped to identify global changes in E. of transcription regulation during bradyzoite
histolytica gene expression related to collagen development (Singh et al 2002).
activation, which in turn, provided a foundation for a
A. C. parvum oligonucleotide microarray was
more comprehensive analysis of Entamoeba
designed to specifically detect Cryptosporidium spp.,
behaviour.
differentiate between the principle genotypes known
MICROARRAY PROGRESS WITH OTHER to infect humans and demonstrated the potential to
PROTOZOAN PARASITES
differentiate between other closely related isolates and
species through single-nucleotide mismatch
As a tool to discover new genes and to identify discrimination (Straub et al 2002).
coordinate expression of family of genes, the
microarray has already proven effective for the In contrast to the apicomplexan microarray reports,
apicomplexan parasites Plasmodium and Toxoplasma studies of kinetoplastids have not advanced
(Duncan, 2004).
significantly beyond validation of the data and
discovery of new genes (Duncan, 2004). A genomic
The earliest malaria microarrays were constructed microarray study of Trypanosoma brucei compared
from 3648 random inserts from a P. falciparum mung gene expression in bloodstream and procyclic
bean nuclease genomic library (Hayward et al 2000). trypanosomes. Hybridizations identified
Through differential hybridization and sequencing of approximately 300 clones that appeared to be
relevant clones, large differences in gene expression differentially expressed (Diehl et al 2002). One study
were identified between the trophozoite and with T. cruzi reported changes in gene expression that
gametocyte stages. A microarray of known P. occur as T. cruzi trypomastigotes convert into
falciparum cDNAs was constructed using expressed amastigotes and identified 38 genes upregulated in
sequence tags harvested at five different stages of amastigotes (Minning et al 2003).
erythrocyte development and revealed common
patterns of expression of groups of genes involved in In the other kinetoplastid genus, Leishmania, several
carbohydrate metabolism, adhesion/invasion and studies in two species have used microarray analysis.
translation machinery (Ben Mamoun et al 2001). A In L. major, a randomly sheared genomic microarray
was used to study the changes in gene expression

14 DNA Microarray in parasitic diseases
JPD : Vol. 28 (1), 2004
among log-phase promastigotes, metacyclic
promastigotes and lesion amastigotes (Beverley et al
2002). The microarray results showed upregulation in
log phase of genes encoding ß-tubulin, histones and
ribosomal proteins, while in metacyclics this included
HASP/gene B, SHERP/geneD, META1 and HSP70.
Similar results were found in comparisons of logphase
promastigotes versus amastigotes. The same
microarray was used to analyze the global changes in
gene expression as procyclic promastigotes
differentiated in vitro into metacyclic promastigotes
(Saxena et al 2003). Using a L. major cDNA array,
changes in transcript abundance for different
developmental stages across L. donovani and L. major
were compared (Almeida et al 2002). This analysis
identified 13.5% unique genes that were significantly
upregulated in amastigotes. Taking the microarray
results, amastigote-expressed genes were. used in
high-throughput DNA-vaccine screens to identify
potential vaccine candidates. Gene expression in L.
donovani was compared between cultured
promastigotes and axenic amastigotes on a genomic
microarray containing 2304 clones (Duncan, 2004).
The results identified many clones from apparently
"new" genes. The major contribution of these studies
was in validation of the array and new gene discovery,
but no pattern of coordinate expression of functionally
regulated or pathway-specific genes was described
(Duncan, 2004).
MICROARRAY PROGRESS WITH
TREMATODE AND NEMATODE PARASITES
The first generation S. mansoni cDNA microarray
consisted of 576 expressed sequence tags selected
from three different cDNA libraries and originating
from two different parasite developmental stages
(Hoffmann et al 2002). The microarray hybridizations
identified 12 novel female-associated and 4 new male-
associated gene transcripts in the mature adult
Schistosome.
cDNA microarray was used to evaluate a discrete
biological event related to the life cycle of Ascaris
suum, a nematode parasite of swine (Morimoto et al
th
2003). Gene expression patterns of A. suum 4 -stage
larvae from the jejunum of infected pigs at 21 days
after inoculation showed that sequences from muscle
actin and myosin, ribosomal protein L11,
glyceraldehyde-3-phosphate dehydrogenase and the
flavoprotein subunit of succinate dehydrogenase were
highly expressed in this larval stage.
Gender-specific gene expression was studied in
Trichostrongylus vitrinus, which is an important
nematode parasite of small ruminants in temperate
climatic zones (Nisbet and Gasser, 2004). Microarray
analysis demonstrated gender-specific expression for
518 of the 716 T. vitrinus expressed sequence tags.
Genes involved in gametogenesis, embryogenesis,
reproduction and different cellular processes were
expressed in a gender-specific manner.
CONCLUSION
Expression profiling by microarray analysis has been
reported from a small number of taxa, and no doubt the
popularity of this technique will increase in
parasitology as resources become more widely
available (Ellis et al 2003). Complete genomic
sequences of different parasites and hosts will offer
sophisticated new strategies for studying host-
pathogen 'interactions. Apart from the construction of
DNA micrqarrays for parasite genome, host profiling
might also identify gene expression signatures unique
for each parasite. Despite the success of DNA
microarrays in gene expression profiling, it is the
activity of encoded proteins that directly manifest
gene function. As protein function is a direct
consequence of the protein product of the gene and as
mRNA levels do not always correlate well with
protein, it is desirable to analyse the entire protein
complement of a cell etc. on a similar scale to mRNA
(Cutler, 2003). In terms of applicability and ease of use
in a microarray format DNA has several advantages
over protein. Still, analysis of protein function and
protein interactions at the genomic scale is becoming a
reality, albeit at the moment restricted to lower
eukaryotes. In the next few years, it is likely that the
use of protein arrays for other organisms, especially
parasites will be available with continued innovation.
Although each technique brings with it both
advantages and limitations, taking the technology of
parasite DNA microarrays, host arrays and protein
arrays into an integrated approach will provide a novel
tool for diagnosis, prognosis, and clinical management
of parasitic diseases.
ACKNOWLEDGEMENTS
Mr. A. Debnath is grateful to UNESCO-American
Society for Microbiology for selecting Short-term
International fellow and providing the travel grant to

JPD : Vol. 28 (1), 2004
DNA Microarray in parasitic diseases
15
work at Sandler Center for Basic Research in Parasitic Cummings CA and Relman DA. 2000. Using DNA microarrays to
Diseases, University of California, San Francisco, CA study host-microbe interactions. Emerging Infectious
Diseases. 6 : 513-525.
94143, USA and The Sandler Family Supporting
Foundation for supporting the E. histolytica Cutler P. 2003. Protein arrays: the current state-of-the-art.
microarray work. Mr A. Debnath and Mr. A. Sen are
Proteomics. 3 : 3-18.
also grateful to ICMR and DBT, Govt. of India Debnath A, Das P, Sajid M and McKerrow JH. 2004. Identification
respectively, for financial assistance provided to them ofgenomic responses to collagen binding by trophozoites of
Entamoeba histolytica. Journal of Infectious Diseases.190: 00
during their stay at-NICED, Kolkata, India. Adam
(in press).
Carrol of the UCSF Genomics and Proteomics Core
provided invaluable aid and discussion Degrave WM, Melville S, Ivens A and Aslett M. 2001. Parasite
genome initiatives. International Journal for Parasitology. 31 :
(http://derisilab.ucsf.edu/core/). 532-536.
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Journal of Parasitic Diseases
Vol. 28 (1) June, 2004, pp. 17-22
Epidemiological studies on bovine microfilariasis in coastal
districts of Andhra Pradesh
V. PAVAN KUMAR, B. SREEDEVI*, T. VENKATA REDDY AND K. NALINI KUMARI
Department of Veterinary Epidemiology and Preventive Medicine,
College of Veterinary Science, Tirupati - 517 502. Andhra Pradesh
In order to record the prevalence of bovine microfilariasis. 2399 blood samples from bovines (2168 buffaloes
and 231 cattle) in the coastal districts (Tanuku, Mandapeta and Gudiwada) and Tirupati were screened
during the period of November 2001 to June 2002-The overall prevalence recorded in bovines was 4.29 per
cent (103 / 2399). The prevalence observed in buffaloes and cattle was 4.43 per cent (96/2168) and 3.03 per
cent (7/231) respectively. Highest prevalence was observed in coastal areas, that is Tanuku (5.61%), followed
by Mandapeta (3.30%) and Gudiwada (3.22%). whereas in Tirupati, the prevalence observed was only 1.92
per cent. Animals of above nine years age group were found to be more prone for the disease (9.54%),
followed by the age groups of six to nine years (4.83%) and three to six years (2.16%), while a lowest
prevalence (0.88%) was recorded in below three years age group. The prevalence noticed in female was 4.32
per cent (103/2382) whereas no positive case could be detected among the 17 male animals screened. With
regards to breed wise analysis, highest prevalence was observed in graded Murrah (5.21%) than nondescriptive
(2.95%) buffaloes. Testing of white cattle for microfilariasis revealed an prevalence of 3.57
(7/196) and nil (0/35) in cross bred and indigenous cattle respectively A higher rate of prevalence of the
disease was noticed in lactating animals (5.46%) followed by non-lactating animals (2.67%) and heifers
(1.53%). No case was recorded among the calves and adult males screened. Highest prevalence of the disease
was noticed during monsoon (3.77%) followed by summer (3.14%) and the least prevalence during winter
(2.11 %).
Key words : Andhra Pradesh, Bovines, Epidemiology, Microfilariasis
I
nfectious diseases have been identified as one of the
major factors disrupting the development of dairy
industry in India. Among them parasitic diseases even
though are not life threatening, jeoparadise the
economy of farmers. Among various parasitic diseases
of livestock, filariasis is an important entity with a
worldwide distribution. Filariasis caused by
nematodes belonging to the filariodea consists of
important genera, which include Setaria, Onchocerca,
Stephana filaria, Parafilaria and Elaeophora. Adult
worms are inhabitants of the peritoneal cavity of the
animals without causing any adverse effects. But the
infective larval stage (L3 form), of these adult worms
known as microfilariae cause a clinical condition
'microfilariasis'. It inflicts economic loss indirectly
due to gradual deterioration in general body condition,
marked decline in milk yield, prolonged convalescent
*Corresponding Author
period and treatment costs. The present investigation
was carried out to study the epidemiology of bovine
micro filariasis including its prevalence with respect to
species, age, breed, sex, physiological status and
season.
MATERIALS AND METHODS
The present study was carried out in West Godavari,
East Godavari, Krishna districts and Tirupati (Chittoor
district) to assess the prevalence of bovine
microfilariasis. The blood samples from all age
groups, both sexes and animals in different
physiological status were collected from buffaloes and
cattle. The samples were screened for microfilariasis.
Blood samples and smears were subjected to different
diagnostic methods namely wet blood film
examination, thick blood smear examination
(Chatterjee, 1981) and modified Knot's method(
Vihan. 1991). Among these three tests modified knot's

18 Epidemiological studies on boine microfilariasis
JPD : Vol. 28 (1), 2004
method and thick blood smear method were found to
be more sensitive. Hence, in this sero epidemiological
survey modified Knot's method was followed.
Staining : For identification of the species of
microfilariae and study of various morphological
structures Giemsa and JSB stainings were followed. The
morphological characterisation of the microfilaria was
done according to the method described by Sloss et al.
(1994). The micro filaria identified in the present study
were presumed to be the larval forms of Seteria cervi.
Study of periodicity : The periodicity pattern of the
microfilaria was studied at every three hrs interval for
a duration of 24 hrs. The peak concentration was
observed at 9 pm. The bovine population presented in
the Veterinary institutions in these areas during the
period of November 2001 to June 2002 were screened
for microfilariasis by different methods of diagnosis.
The details regarding the study population are given in
a tabular form (Table I).
Epidemiological data has been collected with respect
to host, agent and environmental factors. Data was
collected in a prescribed proforma with the following
parameters like place, date, owner's name, case
number, species, age, sex, season, clinical signs, milk
production, agro-climatic conditions (temperature and
relative humidity), geographical information with
respect to surroundings and vector population. The
statistical analysis of the data was carried out by
using the standard methods of Snedecor and
Cochran. (1994).
RESULTS AND DISCUSSION
Area wise prevalence : The area wise prevalence of
the disease observed was 5.61 per cent (63/1123), 3.30
Prevalence : The prevalence of bovine microfilariasis
was studied in Tanuku (West Godavari), Mandapeta
(East Godavari), Gudiwada (Krishna) and Tirupati
(Chittoor). In the present study totally 2399 bovines
were screened. In this 2168 buffaloes and 231 cattle
were screened for microfilariasis between November-
2001 to June-2002. The overall prevalence recorded
was 4.29 per cent (103 / 2399). Similar finding was
reported by Sastry et al. (1985) who recorded an
overall prevalence of 5.25 % in East Godavari district,
Andhra Pradesh.
per cent (10/303), 3.22 per cent (28/869) and 1.92 per
cent (2/104) in Tanuku, Mandapeta, Gudiwada and
Tirupati respectively (Table I). Higher prevalence was
recorded in coastal areas than the dry areas. This may
be due to swampy ness of these areas with a yearlong
high humid climate. Urquhart et al. (1996) reported
that the prevalence of the disease will be more in
warmer areas where there will be long seasonal
activity of the vector population.
Species wise prevalence : Species wise prevalence
noticed was 4.43 per cent (96/2168) and 3.03 per cent
(7/231) in buffaloes and cattle respectively. This
difference in prevalence rates was statistically not
significant (Table 1). Similar prevalence rate was
observed by Sastry el al.. (1985), Satish. (1996) and
Venu et al. (2000).
Age wise prevalence : Analysis of data in the present
study revealed a highest prevalence in the animals of
above nine years (9.54%) of age followed by groups of
six to nine years (4.83%) and then three to six years
(2.16%) age (Table II). Least prevalence was
observed in animals below three years (0.88%) of
age group. This finding is in accordance with the
reports of Kumar et al. (1993) and Sharma and
Kumar (1994) who recorded highest prevalence in
animals aged above 12 years.
Statistically there was no significant difference in the
prevalence between below three years and three to six
years of age groups. However, there was significant
difference (P < 0.01) in the prevalence between below
three years and other age groups (six to nine years and
above nine years). There was significant difference in
the prevalence between three to six and six to nine
years of age at one per cent level and six to nine and
above nine years of age groups at five per cent level.
Breed wise prevalence : Out of 1421 graded Murrah
buffaloes screened for microfilariasis. 74 were found
to be positive with an prevalence of 5.21 per cent
(74/1421). In non-descriptive buffaloes, 22 out of 747
(Table III) were positive for microfilariasis with an
prevalence of 2.95 per cent (22/747). Normal deviate
test for proportions of prevalence between the breeds
revealed Z-value of 2.65 indicating higher prevalence
in graded Murrah buffaloes, which was statistically
significant (P < 0.01). This finding corroborates the

JPD : Vol. 28 (1), 2004
Epidemiological studies on boine microfilariasis
19
Table I : Areawise and specieswise prevalence of bovine microfilariasis
Number of bovines screened for Number of bovines screened for Percentage prevalence
Area microfilariasis microfilariasis Z value
Cattle Buffaloes Total Cattle Buffaloes Total Cattle Buffaloes Total
Tanuku (West Godavari) 102 1021 1123 5 58 63 4.90 5.68 5.61
Mandapeta (East Godavari) 18 285 303 0 10 10 0.00 3.51 3.30
Gudiwada (Krishna) 42 827 869 - 28 28 0.00 3.39 3.22
Tirupati (Chittoor) 69 35 104 2 - 2 2.90 - 1.92
NS NS NS
Total 231 2168 2399 7 96 103 3.03 4.43 4.29 1.15
NS : Not significant
Table II : Agewise prevalence of bovine microfilariasis
Particulars < 3 years 3-6 years 6-9 years > 9 years Z - value
Number of bovines screened for microfilariasis 113 741 1304 241 Z
1.2
= 1.24
NS
Z
1.3
= 3.71**
Number of positives for microfilariasis 1 16 63 23 Z 1.4 = 4.15**
Z 2.3 = 3.35**
Percentage incidence 0.88 2.16 4.83 9.54 Z
2.4
= 3.76**
Z
3.4
= 2.38*
NS : Not significant ** Significant at P < 0.01 * Significant at P < 0.05

20 Epidemiological studies on boine microfilariasis
JPD : Vol. 28 (1), 2004
Table III : Breedwise prevalence of bovine microfilariasis
Buffaloes Cattle
Particulars Graded MurrahNon-Descriptive Total Cross bred Indigenous Total Z-value
(1) (2) (3) (4)
Number of animals screened for microfilariasis 1421 747 2163 196 35 231
Number of positives for microfilariasis 74 22 96 7 0 7 Z
1.2
= 2.65**
Percentage incidence 5.21 2.95 4.43 3.57 - 3.03
** Significant at P < 0.01
Table IV : Sexwise prevalence of bovine microfilariasis
Particulars Females Males Total
Number of bovines screened for microfilariasis 2382 17 2399
Number of positives for microfilariasis 103 - 103
Percentage incidence 4.32 - 4.29

JPD : Vol. 28 (1), 2004
Epidemiological studies on boine microfilariasis
21
Table V : Prevalence of bovine microfilariasis with respect to physiological status
Females Males
Particulars Calves Heifers Lactating Non-lactating Calves Adults Z-value
animals animals
(1) (2) (3) (4) (5) (6)
Number of animals screened for microfilariasis 39 131 1501 711 6 11 Z2.3 = 3.22**
Number of positives for microfilariasis - 2 82 19 - - Z2.4 = 0.93NS
Percentage incidence - 1.53 5.46 2.67 - - Z3.4 = 3.31**
NS : Not significant ** Significant at P < 0.01
Table VI : Seasonwise prevalence of bovine microfilariasis (April 2001 to March 2002)
Particulars No. of bovines screened No. of positives Percentage incidence Z-value
(1) (2) (3)
Summer (March - June) 1719 54 3.14 Z1.2 = 1.10NS
Monsoon (July - October) 2360 89 3.77 Z1.3 = 1.92NS
Winter (November - February) 1850 39 2.11 Z2.3 = 3.23**
NS : Not significant ** Significant at P < 0.01

22 Epidemiological studies on boine microfilariasis
JPD : Vol. 28 (1), 2004
observation made by Satish. (1996) and Kumar et al. rainy season. In contrast. Mukherjee (1965) recorded
(1993) who reported higher prevalence in graded higher prevalence during summer and lowest in rainy
Murrah buffaloes, than non-descriptive buffaloes. seasons.
Out of 196 crossbred cattle screened for
microfilariasis. seven were positive with an
REFERENCES
Chatterjee KD. 1981. Parasitology (Protozoology and
prevalence of 3.57 per cent (7/196). In case of 35 Helminthology) in relation to Clinical Medicine 12th Edn.
indigenous cattle screened, none was found to be Chatterjee Medical Publishers. Calcutta.
positive for microfilariasis.
Kumar M, Joshi HC and Garg SK. 1993. Prevalence of Setciriti
Sex wise prevalence : Among 2382 female animals
cen-i in buffaloes of Kumaon region. Indian Journal of
Veterinary Research. 2 : 1-6.
screened for bovine microfilariasis 103 were found to
Mukherjee RP. 1965. Percentage of microfilarial infections and the
be positive with an prevalence of 4.32 per cent period of their maximum concentration in the blood of
(103/2382). whereas out of 17 male animals screened, buffaloes. Indian Veterinary Journal. 42 : 131-133.
none were positive (Table IV). However, no Sastry GJS, Rao VP, Narsimham MVVL and Rao PK. 1985.
conclusion could be drawn on sex wise prevalence due Animal filariasis in East Godavari district, Andhra Pradesh.
to small sample size.
Livestock Adviser. 10: 56-60.
Prevalence with respect to physiological status :
The observations pertaining to the prevalence of
microfilariasis in this study showed highest
prevalence in lactating animals (5.46%) followed by
non-lactating animals (2.67%) and heifers (1.53%).
No case was recorded in calves and adult male
buffaloes (Table V). The normal deviate test for
proportions of prevalence between heifers and
lactating animals: between lactating and non-lactating
animals showed a significant difference (P < 0.01)
indicating highest prevalence in lactating animals.
However, there was no significant difference in the
prevalence between heifers and non-lactating animals.
Satish Udhav, Rao Digrasakar. 1996. Clinico therapeutic studies
on Microfilariasis in Buffaloes (Biihualus huhulis). Ph.D.
Thesis. Acharya NG Ranga Agricutlural University,
Rajendranagar. Hyderabad.
Sharma MC. 1985. Patho-biochemical and therapeutic studies on
filariasis in cattle, buffaloes and dogs Ph.D. Thesis. G B Pant
University of Agriculture and Technology. Pantnagar.
Sharma SP and Kumar M. 1994. Studies on the prevalence of
clinical Setaria infection in buffaloes and horses. Indian
Veterinary Journal. 71: 1243-1245.
Sloss MW, Kemp RL and Zajac AM. 1994. Veterinary Clinical
Parasitology 6th Edn. Iowa State University Press. Ames. India,
Snedecor GW and Cochran WG. 1994. Stalisficul methods. 8th
Edn. Oxford and IBH Pub. Co. Pvt.Ltd. Calcutta.
Urquhart GM. Armour J. Duncan JL. Dunn AM and Jennings FW.
Season wise prevalence : The yearlong data (April 1996. Veterinary Parasitology lind Edn. Blackwell Science Ltd.
2001 to March 2002) collected from the veterinary
Venu R. Radhakrishna Murthy P and Sreedevi C. 2000. Prevalence
institutions (Tanuku, Mandapeta and Gudiwada) was
ofmicrofilariasis in graded Murrah buffaloes in West Godavari
subjected to analysis for studying the prevalence of district of Andhra Pradesh. Indian Veterinary Journal. 77: 272-
microfilariasis with respect to season. The highest 273.
prevalence (3.77%) was recorded during monsoon Vihan VS. 1991. Modern Veterinary Laboratory Techniques in
(July - October). The prevalence in summer (March -
June) was next to monsoon showing percentage
prevalence of 3.14 (Table VI). Least prevalence
(2.118%) was observed during winter (November -
February). Statistically however, there was no
significant difference in prevalence during summer
and other seasons (i.e. monsoon and winter). whereas
there was significant difference (P < 0.01) in the
prevalence during monsoon and Winter. Sharma
(1985) and Satish. (1996) reported peak prevalence in
Clinical Diagnosis 1st Edn. CBS Publishers and Distributors.
Delhi.

Journal of Parasitic Diseases
Vol. 28 (1) June 2004, pp. 23-28
Seasonal occurrence of helminth parasites in Schizothorax
in Dal Lake Kashmir
A. R. KHAN*, M. Z. CHISHTI, FAYAZ AHMAD, MAJIDAH RASHID & SHAFQAT BAKSHI
Post Graduate Department of Zoology, University of Kashmir, Srinagar, India: 190 006
Helminth parasitization is one of the major factors responsible for the decline of fish fauna of the valley, which
provides protein to almost 60% of the people of valley in addition to providing sport and employment to Parge
population particularly in rural areas. The present paper is an attempt to highlight the occurrence of different
helminth groups in different seasons in order to find out a relationship between the helminth occurrence and
season supported by data spreading over more than 3 years from 1991-93 and again from 1997-1998 In all four
helminth groups including Trematoda, Cestoda, Nematoda and Acanthocephala infecting the native fish-
Schizothorax, were studied represented in the present studies by 9 genera including Diplozoon, Clinostomum
and Allocreadium from Trematoda; Adenoscolex from Cestoda; Pomphorhynchus, Ncoechinorhyncus and
Tenuiproboscis from Acanthocephala and Rhabdochona from Nematoda group. About 610 Schizothorax
specimens from Dal lake were studied through regular periodical collections. The hosts were scanned for
helminth presence and the data analysed for seasonal occurrence of the helminths. The tables (1-4) clearly
depict the seasonal occurrence of helminth parasites indicating that season plays a vital role in the fish
parasitization.
Key Words: Fish; Helminths; Seasonality.
INTRODUCTION
Total number of hosts infected
Prevalence = --------------------------------- xl00
he population of Schizothorax is declining in the
Twaters of Kashmir. The reasons may be varied, but
Total number of hosts examined
helminth infestation is an important factor responsible
for this decline. In order to conserve this native fish,
Total number of Parasites
studies from all corners is important and the present
Mean intensity = ------------------------------------------
study deals with .the occurrence of helminth parasites in
Total number of hosts infected
different seasons in an urban lake-The Dal lake of
Total number of parasites
Kashmir. Relative density = -----------------------------------------
or Abundance Total number of hosts examined
MATERIALS & METHODS :
Five hosts belonging to the native genus Schizothorax
were collected alive fresh fortnightly from fishermen in
Dal Lake & analysed in the laboratory through the
routine Parasitological techniques. The following
ecological terms as given by Morgoll's et al (1982) are
used in the present study.
* Corresponding Author
OBSERVATION
Schizothorax was sampled throughout the three years in
Dal lake and a total of 600 specimen were investigated
for helminlhological survey. The occurrence of
different helminth parasites in the four seasons was
studied. The helminthological patterns of this host
observed parasite-wise in different seasons were as
follows :

24 Seasonal occurrence of helminth parasities in fish
JPD : Vol. 28 (1), 2004
Trematoda - Diplozoon
44 Acanthocephalan parasites belonging to the genus
Pomphorhynchus. The infection again showed a
Out of the three trematode genera viz. Diplozoon,
seasonal occurrence, as the fishes were found to be
Clinostomum and Allocreadium recorded during the
infected either during spring or early summer, while for
present investigation in different fish hosts, the rest of the survey period no infection of the parasite was
infection of the only ectoparasitic genus Diplozoon was
recorded as shown in table III.
recorded in this host from Dal lake. Whereas
Clinostomum and Allocreadium were absent. The fishes The prevalence of infection of Pomphorhynchus was
were invariably found to be infected with the Diplozoon highest i.e. 5. 74 during spring and the minimum of 1. 14
parasite i.e. Diplozoon kashmirensis, although M. 1. during summer season. Minimum M. 1. of 1.5 during
and R. D. showed variations as shown in the table I. summer and maximum M. 1 (3. 41) during spring.
Whereas the R. D. of 0.196 was observed during spring
The prevalence of infection of D, Kashmirensis in
and 0. 017 during summer.
Schizotliornx was the highest (19.61) during spring,
while as the minimum value of prevalence was recorded Nematoda - Rhabdochona
in the autumn season (i.e. 9.70) . The highest M. I. of
3.46 was recorded during autumn and the minimum of
The infection of Nemaloda in Schizothorax. from Dal
1.92 during summer. Maximum R.D. of 0.55 was
Lake during tlie present 3 years of investigation was
observed in spring season and minimum of 0.29 in
restricted to the spring season. While for rest of the
summer season .
season of the year the infection was altogether absent.
Cestoda - Adenoscolex oreini
The prevalence of Rhabdochona was the minimum out
of the four groups of parasites recorded so far and
Out of the total of 610 fishes from Dal Lake only five restricted to a short period of late spring, the prevalence
fishes were found to be infected with 9 parasites being 1.91 in spring, M. I. being 2 and R. D. 0. 03 as
belonging to the cestode genus Adenoscolex . Cestode shown in table IV.
infection showed a marked seasonal occurrence as the
infection was observed only during spring and summer
DISCUSSION
seasons as is clear from the Table II.
Studies on seasonal dynamics of fish parasites with respect
The prevalence of infection of Adenoscolex being 1.43
to the fluctuations of the parasitic fauna are meager.
during spring; and 1.14 during summer. M. 1. of
Williams (1963, 1979), Chubbs and Thomas (1964).
infection was 2.5 during summer season and 1.33
Kennedy (1969), Tedia (1970), Hare and Burt (1974-75),
during spring R. D of 0.02 during summer and 0. 019
and Cling (1988), Carried out studies on the seasonal
during spring.
influence on the helminth parasites and concluded that,
season plays a significant role in the prevalence and
Acanthocephala - Pomphorhynchus
intensity of helminth parasitization of fish.
A total number of 14 Schizothorax out of 610 fishes
from Dal Lake dissected were found to be infected with
In the present study following results on four helminth
groups were arrived at :
Table I: Seasonal Changes of Infection pattern of D. kashmirensis
Seasons No. of No. of %age No. of M. I. R.D.
fishes fishes infected parasites
exam. infected
Summer 175 27 15.42 52.0 1.92 0.29
Autumn 134 13 9.70 45 3.46 0.33
Winter 92 14 15.21 41 2.92 0.44
Spring 209 41 19.61 115 2.80 0.55

JPD : Vol. 28 (1), 2004
Seasonal occurrence of helminth parasities in fish
25
Trematoda
From this group the ectoparasitic monogenetic
trematode Diplozoon was found to be infecting this fish
host genus, although the occurrence and intensity
fluctuated considerably. Evans (1978) while studying
the trematodes, observed that the prevalence and
intensity of infection increased during the spring and
summer and by July and August the majority of
parasites were also fully mature.
A marked seasonal effect was noted, being most
abundant during the summer on the gill parasites of
Lcpomis, by Ilanck (1978), Meyers (1978) and Dronen
(1978).
Glenn (1980) observed that the prevalence of infection
of Crepidostomum hiodontis (Trematode) increased in
August and September to a relatively high constant
winter rate of approximately 86%. This tremalode was a
common parasite of both age groups of moon eye. For
the older age C. hiodontis was found during each month,
prevalence was lost in June and July, increased in
August and was relatively constant during the autumn
and winter. But Palurorhynchus hiodntis in infected fish
varied from peaks in summer to lows in winter.
Table II: Seasonal Changes of Infection pattern of Adenoscolex
Seasons No. of No. of %age No. of M. I. R. D
fishes fishes infected parasites
exam. infected
Summer 175 2 1.14 5 2.5 0.02
Autumn 134 - - - - -
Winter 92 - - - - -
Spring 209 3 1.43 4 1.33 0.019
Table III: Seasonal Changes of Infection pattern of Pomphorhynchus
Seasons No. of No. of %age No. of M. I. R. D.
fishes fishes infected parasites
exam. infected
Summer 175 2 1.14 3 1.5 0.017
Autumn 134 - - - - -
Winter 92 - - - - --
Spring 209 12 5.74 41 3.41 0.196
Table IV: Seasonal Changes of Infection pattern o f Rhabdochona
Seasons No. of . No. of %age No. of M. I. R. D
fishes fishes infected parasites
exam.
infected
Summer 175 - - - - -
Autumn 134 - - - - -
Winter 92 - - - - -
Spring 209 4 1,91 8 2 0.03

26 Seasonal occurrence of helminth parasities in fish
JPD : Vol. 28 (1), 2004
Muzzal (1980) while working on the trematode
Triganodistomum attenuatum, recorded peak in spring
season.
Recruitment of Uvulifer ambloplitis by caged blue gill
began in May and ended in September, with the
maximum recruitment occurring in July as reported by
Dennis and Gerald (1984),while working on population
biology of trematodes.
Dennis and Esch (1985) after working on the
trematodes observed peak in July (early summer). They
also supported the significant role of season in the
infection of helminth parasites.
Rand and Burlt (1985) supported the role of season in
the infection of parasites while working on the seasonal
occurrence, recruitment of Allocreadium lobatum
showing that the prevalence of infection increasing to
high levels from November through May.
According to James (1988) size of the host plays a great
role in the infection of parasites as large hosts carried
significantly more Eirocotyle spinicirrus individual
than smaller ones and highest infection for larger
individuals was observed in May, where as the smaller
length classes were dominant from June through
October 1986.
definitive host-lhc dace Leuciscus leuciscus. Infection
of fish is rcslriclcd to colder winter months and the
parasite matures in late spring and winter before
disappearance in summer. Kennedy and Walker (1969)
have shown experimentally that at low temperature C.
laticeps is better able to establish itself in the Dace and
survives for longer periods than at high temperature.
Thus supporting the results arrived during the present
investigation.
Fischer and Freeman (1969) reported the incidence of
enteric adult Proteocephalus ambloplitis in small
mouth bass (Micropterus dolomievi) was highest in
spring declining in the summer and disappearing in late
autumn.
After studying the trcmalodes Gyrodactylus cole-
manensis , G. salmonis by Cone and Cusack (1988), it
was observed that the intensity of infection during
winter to a spring peak followed by a decrease during
the summer months.
The prevalence and mean intensity of Glaridacris
catostomi and Glaridacris larvrei in Oyster river hosts
was highest in spring (1975-76) and winter (1976-77).
The mean intensity of G. castostomic in Bellamy river
hosts was highest in spring 1975 and 1976 as reported
by Muzzall(1980).
Infra population of Adenoscolcx Oreni increased in
winter season as reported by Dhar and Peerzada (1992),
while working on the seasonal variation in the
occurrence and maturation of Adenoscolex Oren
Fotedar 1958 infecting some Cyprinid fishes ofWular
lake (Kashmir) Hence the present observations arc in
conformity with Kennedy (1969), Muzzall (1980) and
Dhar and Peerzada (1992) and Fisher and Freeman
(1969) indicating that season plays a great role in
helminth infections.
The distribution of four species of Dactylogyrus on the Acanthocephala
bream's gill is fairly similar and peak was in spring and
The Acanthocephala group represented by two genera
summer as reported by Dzika and Szymanki (1989).
viz., Pomphorhynchus & Neoechinorhynchus again
Thus the present observations are in accordance with showed a marked seasonal occurrence, as the peak
those of Evans (1978), Rand & Burt (1985), Muzzall infection in all the recorded observations was in spring
(1980), Cones & Cusack (1988), James (1988), While it or early summer season.
shows variation with the work of Hanek (1978), Meyers
The effect of temperature and other factors upon the
(1978), Dennis and Esch (1985).
establishment and survival of Pomphorhynchus laevis
Cestoda
in gold fish was studied by Kennedy (1972). He further
observed that although the density of infection had no
The only ceslodc genus infecting the fish hosts under effect upon the establishment of the parasite but a 12°C
discussion includes the Caryophyllidean genus rise on water temperature resulted in 30% reduction of
Adenoscolex. A seasonal occurrence of this cestode was infection. But the establishment of fish Acanthocephala
observed with two infection period spring and summer is affected by temperature to a lesser extent than that of
in Dal Lake.
some fish cestodes. The degree of infection of various
hosts with Acanthocephala parksidei and the
Kennedy (1969) reported that Caryophyllaeus laticeps
development stages of these Acanthocephala showed
exhibits a well defined seasonal incidence cycle in its

JPD : Vol. 28 (1), 2004
Seasonal occurrence of helminth parasities in fish
27
considerable seasonal variations. Huzinga (1980) and Arnin (1988).
Frequency of infection and maturation was highest
during the spring as reported by Amin (1975), which
clearly supports the present observations.
Muzzall and Bullock (1978) have shown that the
prevalence of Neoechinorhynchus saginalus was high
(above 44% in all months sampled) and fluctuated
irregularly throughout, the sampling period in 1975-76,
the prevalence declined from high in June to a low in
January and March and exhibited a tendency to increase
again towards July. The Prevalence of Acanthocephala
dirus in the Creek Chubs and Cystacanths in isopods
increased through the autumn and winter, peaked in
Feb. 1976 (Isopod) and April 1976 (Chubs) as reported
by Camp and Huizinga (1980) al'lcr studying the
seasonal population interaction of A . dirus.
Nematoda
Nematode infection again showed seasonal occurrence
showing peak availability towards the winter and spring
season.
The data analysed by Stromberg and Crites (1975)
shows seasonal cycle in population structure, site
selection, intensity of infection maturation and
reproduction. Infection of Camallanus oxycephalus
occurs during July and August with a resulting peak in
population density during late summer and autumn.
They further concluded that the infection was generally
more frequent and higher intensity in longer fish.
Abundance of Cystidicoloides tenuissima in the
infected fish varied from peaks in summer to lows in
Muzzall (1980) studying the impact of season on
winter, as worked out by Glenn (1980). C. tenuissima,
prevalence and intensity of helminths concluded that
the most common and abundant species in trout did not
the mean intensity of Pomphorhynchus bulbocolli was
exhibit a pronounced seasonal pattern in prevalence;
highest in spring and summer 1975, spring 1976, and
mean intensity, however, was highest in July 1982, 83 in
winter 1976-77 while as Neoechinorhynchus cristatus
both trout species. The intensity of C. tenuissima
did not exhibit a seasonal cycle in maturation or in
increased as trout become older and than decreased in
length distribution.
brook and brown trout 3 & 4 years of age respectively.
The prevalence and mean intensity of Spinitectus
The Neoechinorhynchus prolixoidcs studied were gracilis were highest in July 1982, 83 & 84.
mostly recruited during late summer early autumn,
reached sexual maturity by spring and continued its
simultaneous reproductive activity and growth through
the summer into the following autumn (Amin, 1986).
He also observed the recruitment of N. Cylindratus in
late summer and autumn. He further noted that the
seasonal periodicity of Neoechinorhychus prolixoides
bore basic similarities to that of N. cylindratus except
that the number of juveniles was more, particularly in
the spring.
Amin (1987) observed that the intensity of infection
increased from low in the autumn to higher levels in
warmer months and reached a maximum usually in the
summer after studying the ecology and host relationship
of Pomiphorhynchus bulbocolli. Amin (1988) reported
Leptorhynchoides thecatus from Micropteruus
salmoidcs. He observed that the egg presence, worm
recruitment and maturation appear to occur throughout
the year with maximum recruitment in the autumn,
maturity and reproduction in spring and summer.
After studying the seasonality of infection and
histopathology of Cystidicoides ephemeridarum, the
researcher Green Wood and Bakcr (1987) observed that
the mean intensity followed a typical seasonal trend in
salmonids. In both years it increased from May to reach
a maximum in June and then decline considerably in
July and August. They also concluded that the season
played a significant role in the intensity of infection.
Thus the present observations show variations with
Stromberg and Crites (1975), Glcnn (1980) and
Greenwood and Baker (1987).
CONCLUSION
From the above account it is clear that season plays a
vital role in helminth infestations although the effect is
different on different helminth groups which may be
related to their life cycle patterns e.g. recruitment,
intermediate hosts etc. Inspitc of resemblances of the
present observations with some previous researchers,
there is a noticeable variation with others. The variation
Thus the present observations are supported by Amin may be due to the different water body, host, feeding
(1975, 1986 and 1987); Muzzall and Bullock (1978); habits of the host, diversity of the climatic conditions
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
JPD : Vol. 28 (1), 2004
study of life cycle in these parasites and the detailed James, E. Joy, 1988 Monthly length class frequencies of Microcotyle
study under experimental conditions in order to find out spinicirrus (Monogenea : Microcotylidae) from the freshwater
drum, Aplodinotus grunniens, in west Virginia. Proc.
the possible role of a particular factor in the recruitment Helminthol. Soc. Wash., 55 (2) : 246-251.
and maturation of a particular parasite
Kennedy, C.R., 1969 Seasonal incidence and development of the
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parksidei Amin, 1974 (Acanthocephala: Echinorhynorhynchidae) in Kennedy, C.R., 1972 The effect of the temperature and other factors
Wisconsin fishes. J. Parasitol., 61 (2): 318-329.
up to the establishment and survival of Pomphorhynchus laevis
(Acanthocephala) in gold Fish, Carassius suratus. J.
Amin, O.M. 1986, Acanthocephala from lake fishes in Wisconsin Parasitology, 65 : 283-294.
host and seasonal distribution of species of the genus
Neoechinorhynchus Hamann, 118.
Kennedy, C.R. and Walker, P.J., 1969 Evidence for an immune
response by dace Leucisucus leuciscus, to infection by the
Amin, O.M. 1987, Acanthocephala from lake fishes Wisconsin cestode Caryophyllaeus laticeps. J. Parasitology, 55 :579-582.
(USA) : Ecology and host relationship of Pomphorhynchus
bulbocoli (Pomphorhynehidae). J. Parasitol., 73 (2): 278-289. Meyer, T.R., 1978 Prevalence of fish parasitism in Raritan Bay, New
Jersey. Proc. Helminthol. Soc. Wash.,45 (1): 120-128.
Camp, J.W. Jr and Harry, W Hvizinga 1980 Seasonal population
interaction of Acanthocephalus dirus (Van cleave, 1931) in the Margolis et al., 1982 The use of ecological terms in Parasitology
Creek Chubb, Semotilus astromaculatus and Isopod, Asellus (Report of an adhoc committee of the American Society of
intermadius, J. Parasitol,. 66 (2) : 299-304.
Parasitologists). J. Parasitol., 68 : 131-133.
Ching, H.L. 1988 The distribution of Plerocercoides of Muzzall, P.M., 1980a Ecology and seasonal abundance of three
Diphyllobothrium dendriticum (Nitzsch) in Sockeye Salmon Acanthocephalan species infecting white suckers in SE New
(Onchorhynchus nerka) smotts from great central lake, British Hemsphire. J. Parasitol., 66 (1) 127-133.
Columbia. Can. J. Zool., 66 : 850-852.
Muzzall, P.M., 1980b Population biology and host-parasite
Cone, D.K. and Cusack R, 1988 A study of Gyrodactylus colemanensis relationship of Triganodistomum attenuatum (Trematoda :
Mizelle and Krilsky, 1967 and Gyrodactylus salmonmis Yin and Lissorchiidae) infecting the winter sucker, Catostomus
Sproston, 1948 (Monogenca) Parasitizing captive salmonids in commersoni (Lacepede). J. Parasitol., 66 (2) 293-298.
Wovw Scotia (Canada) Can. J. Zool, 66 (2): 409-415.
Muzzall, P.M., 1980c Seasonal distribution and ecology of three
Dhar, R.L. and Peerzada, M.Y. 1992 Seasonal variation in the occurrence Caryophyllac : Cestoda species infecting white suckers in SE
and maturation of Adenoscolex Oceni Fotedar, 1958 (Cestoda : new Hemisphere. J Parasitol., 66 (3) : 542-550.
Carophyllaeidae) infecting some Cyprinid fishes of Wular Lake Muzzall, P.M. and Wilburl Bullock, 1978 Seasonal abundance and
(Kashmir). Current Trends in Fish and Fishery Biology and Aquatic host-parasite relationships Neoechinorhynchus saginatusi Van
Ecology (Ed. Yousuf, Raina & Qadri) xv + 365.
Cleave and Bangham, 1949 in the fall fish, Semotilus corporalis
Dronen, Norman O., Jr., 1978 Host Parasite population dynamics of (Mitchill). J. Parasitol., 64 (5): 860-865.
Haematolaechus (Digenea). Am. Midl. Nat., 99 (2) : 330-349. Rand, T.G. and M.D.B. Burt, 1985 Seasonal occurrence recruitment
Dzika, E. and Stanislaw Szymanski, 1989 Co-occurrence and and maturation of Allocreadium lobatum Wallin, 1904 (Digenea :
distribution of Monogenea of the genus Dactylogyrus on gills of Allocreadiidae) in the fall fish, Semotilus corporalis Mitchell, in a
the bream, Abramis brama Acta, Parasitol, Pol., 34(1): 1-14.
new brunswick, Canada Lake system. Can. J. Zool., 63: 612-616.
Evans, N.A., 1978 The occurrence and life history of Asymphylodora Stromberg, P.C. and Crites, J.L., 1975 Population Biology of
kubanicum (Platyhelminthes : Digenea : Monorchidae) in the Cammallanus oxycephalus Ward and Margath, 1916 (Nematoda
Worcester - Birmingham canal, with special habits of definitive host, Cammallanidae) in white bass in western lake. J. Parasitol., 61 :
Rutilus rutilus. J. Zool. Lond., 184 : 143-153.
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Glenn, C.L., 1980 Seasonal parasitic infections in moo eye, Tadla, S. and Fcrnando, C., II. 1970 Observations on seasonal
Hiodon tergisus (Le Sueur), from the Assiniboine River. changes of the parasites of yellow perch from the Bay of Quinte,
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Lake Ontario. Res. Board Can., 26 : 833-843.
Greenwood, S.J. & Baker M.R., 1987 Cystidicoloedes Williams Dennis, D., 1979 Seasonal incidence of Isoglariducris
wisconsincensis in its fish host (Cestoda : Caryophyllidac).
ephermmeridarum (Linstow, 1872) (Nematoda) in speckled
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Can. J. Zool., 65 (II): 2589-2593.
Hanek, G. and CH Fernando, 1978 Seasonal dynamics and spatial
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Lepomis gibbosus (L). Can. J. Zool., 56 : 1241-1243.
IOWA Stale J. Res., 53 (4) : 305-310.
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and G. catastomi in Red cedar river, State J. Res., 53(4): 311-3 16.

Journal of Parasitic Diseases
Vol. 28 (1) June 2004, pp. 29-36
A Monoclonal Antibody to 120 kDA B.Malayi Antigen with
Diagnostic Potential in Bancroftian Filariasis
1 1
BALAJI GANESH. B, PARAB P.B. , KATDARE. M , REDDY MVR AND HARINATH BC*
Department of Biochemistry, JB Tropical Disease Research Centre, MGIMS, Sevagram, Wardha 442 102
1
& National Centre Cell sciences, Pune University Campus, Pune-411 007
A monoclonal antibody (Bm Ab 120) of IgM isotype against B.malayi micro filarial soluble antigen [Bmmf
(S)] that specifically recognized a 120 kDa antigen present in microfilarial soluble extracts, microfilarial
excretory- secretory proteins and adult soluble proteins but not in soluble infective larval extracts was
produced. Bm Ab 120 was employed in a sandwich ELISA to screen individuals in areas endemic for
bancroftian filariasis as well as those suffering from other parasitic infections from non-endemic area using
two different sources of polyclonal antibodies viz, Filarial serum immunoglobulins (FSIgG) isolated from
clinical patients and anti-Bmmf (S) antibodies raised in rabbits as captured antibodies. FSIgG- Bm Ab 120
combination showed 84% mf carriers, 66% of patients with clinical symptoms of filariasis and 18% of
endemic normals as positive for filarial antigens. When anti-Bmmf (S) antibodies were used as capture
antibodies along with Bm Ab 120 antibodies as tracer probe, 88% of mf carriers were detected as also 54% of
clinical cases and 12% endemic normals. A small percentage of patients with Tropical Pulmonary
Eosinophilia were also picked up by both the assays. None of the sera of other parasitic infections such as
ascaris, hookworm, leishmania and malaria showed levels above the cut-off suggesting high specificity of the
monoclonal antibody. Thus, Bm Ab 120 was found to be useful in identifying significant numbers of
asymptomatic microfilaraemics, along with patients suffering form clinical and occult filariasis in
bancroftian endemic areas.
Keywords : Brugia malayi, Diagnosis, ELISA, Filariasis, Monoclonal antibodies, Wuchereria bancrofti,
INTRODUCTION
ymphatic filariasis is a cause of severe morbidity
Lfor people living in filarial endemic areas with an
estimated 120 million people suffering from this
disease worldwide (WHO, 1994). It is caused by the
blood- borne nematodes Wuchereria bancrofti, Brugia
malayi and Brugia timori of which more than 90% of
the cases are caused by W. bancrofti. The World Health
Organization (WHO) has made it a goal to interrupt
transmission and eliminate this disease globally by the
year 2020 (WHO. 1993). The successful
implementation of this ambitious programme would
require the identification and treatment of individuals
harbouring the infection. Besides microfilarial
* Corresponding Author
carriers, clinical and occult filarial patients (showing
atypical manifestations) who are usually
amicrofllaraemic but may harbour parasites in the
internal organs or tissues would also be important
targets for identification and treatment in the
successful implementation of filaria control
programmes.
Specific monoclonal antibodies produced against
antigens of mammalian stages of filarial parasites
would act as suitable diagnostic agents for screening
and identification of target population and could also
have epidemiological implications. Lack of suitable
animal models to maintain W. bancrofti infection has
hampered availability of sufficient parasite material
due to which heterologous antigens from other
nematodes have been exploited to develop serological

30 Monoclonal antibody Bm Ab120 in diagnosis of filariasis JPD : Vol. 28 (1), 2004
tests. Currently available diagnostic tools for antigen peritoneal lavage of infected jirds or at necropsy.
detection include the ICT Filariasis and the Trop Bio Worms were first lyophilized, homogenized,
ELISA that employ monoclonal antibodies directed sonicated and antigens extracted in PBS overnight.
against Dirofilaria immitis and Onchocerca gibsoni The concentrated antigen was labeled as A (S) antigen.
(both animal parasites) respectively and show high
Immunization of mice : Six week old BALB/C mice
sensitivity in detecting microfilarial carriers
were immunized with 100 micro grams of Bmmf (S)
(Chandrasena et al, 2002; Ramzy et al, 1999) but not
antigen intra-peritoneally (i.p) on days 1, 8, 15 and 22.
always in communities showing clinical
The first immunization was with equal volume of
manifestations of disease (Eigege et al, 2003; Nguyen nd rd
Freunds complete adjuvant while the 2 and 3 doses
et al, 1999). However, individuals showing prepatent
th
were with Freunds Incomplete adjuvant. The 4 and
infection and mild or atypical manifestations of
final dose was without any adjuvant. Animals that
filariasis but absence of microfilaria in peripheral
developed high antibody litres to Bmmf (S) as
blood may escape detection by these assays. Diagnosis
revealed by ELISA were used as donors of spleen cells
of filarial etiology and morbidity control is equally
for the production of hybridoma.
important in filaria control programme. We therefore
explored to develop a monoclonal antibody against Production of monoclonal antibodies (mAbs) : Cell
microfilarial antigens of the closely related human fusion was performed according to the method
nematode, B. malayi, and evaluate the presence of described earlier (Galfre et a/, 1977), with minor
these antigens in a spectrum of population living in an modifications. Briefly, spleen cells isolated from mice
area endemic for bancroftian filariasis.
immunized as above and SP2/0 myeloma cells
MATERIAL AND METHODS
(National Centre for Cell Science, Pune, India) were
washed twice in serum-free Dulbecco's Modified
Antigen preparations: Brugia malayi microfilariae Eagle Medium (DMEM, Gibco-BRL). The spleen
were collected by the peritoneal lavage of B.malayi cells were mixed with myeloma cells at a ratio of 1:1
infected jirds. The microfilariae were plated on sterile and were pelleted together at 400g for 5 min. Cell
petri plates in order to allow the host peritoneal cells to fusion was promoted by the use of polyethylene glycol
settle. They were then maintained in vitro as described (PEG-1500, BDH chemicals, Ltd). The fused cell
earlier (Alli et al., 2000) to prepare B.malayi
6
pellet was suspended to a concentration of 1 X 10
microfilarial excretory-secretory antigens (Bm mf cells/ 0.5 ml in DMEM containing 10 % fetal calf
ES). The parasites were then separated by serum and hypoxanthine, aminopterin and thymidine
centrifugation, homogenized in ice in the presence of (HAT, Sigma Chemicals). This, suspension was
cocktail protease inhibitors [0.1 mM ethylene diamine dispensed into 24- well flat-bottomed culture plates
tetraacetic acid (EDTA) and Tosyl-L-lysine (0.5 ml/well) already containing mouse peritoneal
chloromethyl ketone (TLCK)], sonicated and macrophages as a feeder layer and were incubated at
extracted overnight at 4°C in 0.05M PBS and the 37°C in a humid atmosphere at 5% CO 2 in air. Culture
homogenate was centrifuged at 12,000 rpm for 20 supernatants from wells displaying dense turbid cell
minutes at 4°C. The supernatant was removed, growth were tested for the presence of antibody to
concentrated by ultra membrane filtration and labeled Bmmf (S) by ELISA. Antibody-producing hybrids
as Brugia malayi microfilarial soluble antigen were then cloned by a serial dilution technique on a
[Bmmf(S)Ag].
feeder layer of mouse peritoneal macrophages.
B.malayi infective larvae (L3) obtained by the Positive clones were grown as ascites tumors in mice.
dissection of infected mosquitoes were treated Purification of mAbs : Antibody producing cells
similarly and the proteins obtained labeled as L3 (S) from isolated clones were propagated in vivo as ascites
antigen. Brugia malayi adult worms were collected by in pristane- treated mice. SP2/0 myeloma cells were

JPD : Vol. 28 (1), 2004
Monoclonal antibody Bm Ab120 in diagnosis of filariasis
31
injected similarly into mice for production of to check for cross-reactivity.
irrelevant antibody for use in control experiments.
Indirect ELISA: This technique was employed to
Ascitic fluid was harvested from experimental and
screen culture supernatant for antigen-specific
control mice and centrifuged to remove cells.
hybridomas and to check reactivity of mAb with
Immunoglobulins (Ig) were isolated by ammonium different stages of B.malayi. Briefly, polystyrene
sulphate precipitation as described earlier plates (Nunc) were coated with 100 micro litres (5
(Subrahmanyam et al.,1990').
micro grams /ml) of Bmmf (S)/ A(S) / L3(S) antigen in
sodium carbonate-bicarbonate buffer(pH 9.6) and
Polyclonal antibodies : Filarial serum
incubated at 4° C overnight. Plates were drained and
immunoglobulins (FSIgG ) from serum of clinical
incubated in 0.5% gelatin in PBS for 1h at 37°C. Plates
patients were isolated by ammonium sulphate
were then washed in PBS-Tween 20 (PBS/T) once.
precipitation followed by ion-exchange
chromatography on DEAE column as described earlier One hundred micro litres of hybridoma supernatants /
(Alikhan et al, 1990) while anti-Bmmf (S) antibodies Bm Abl20 were then added and incubated at 37°C for
were isolated from rabbits immunized with Bmmf (S) 1h. After 3 washes, wells were incubated with 100
antigen similarly.
micro litres of goat anti-mouse peroxidase conjugate
and incubated for 1h at 37°C, washed 5 times and
Human sera: Blood samples were collected from
incubated with 100 micro litres of ABTS-H2O
2
villages surrounding Sevagram, Maharashtra, India
that are highly endemic for bancroftian filariasis and
substrate. The colour developed was read at 415nm.
from patients visiting the out-patient department of Sandwich ELISA: This was done to screen sera of
Kasturba Hospital, Sevagram with clinical symptoms endemic population with monoclonal antibody for
of filariasis such as lymphoedema, hydrocele, specific antigen.. Sandwich ELISA was performed by
elephantiasis, lymphadenitis and epididymoorchitis. coating 5 micro grams /ml of anti-Bmmf (S) or FSIgG
The patients were classified into Grades I, II and III antibodies per well and incubating overnight at 4°C.
depending upon their clinical symptoms as described Plates were washed once with PBS/T and blocked in
earlier (Alikhan et al., 1990). Blood samples were also 1% BSA (Sigma) for 1 h at 37°C. Plates were washed
collected from patients suffering from atypical again twice and incubated with 100 micro litres serum
manifestations of filariasis such as Tropical (1:100 in PBS/T) for 1h at 37°C. After 5 washes, plates
Pulmonary Eosinophilia (TPE), Urticaria and were incubated with 100 micro litres monoclonal
bronchial asthma of suspected filarial etiology and antibody (1 micro gram/ml) for 1h at 37°C followed by
grouped under 'Occult' filariasis. Twenty five of the 5 more washes and incubation with 100 micro litres of
sera in this group comprised of TPE sera, 10 bronchial 1:2000 diluted goat anti-mouse HRPO for 1 h at 37°C.
asthma cases and 5 cases of urticaria. Microfilaraemic Three mg of 2,2'- Azino- di (3-ethyl) benzthiazoline
carriers were detected by presence of microfilariae on sulphonic acid (ABTS) in 10ml of citrate - phosphate
microscopic examination of night blood smear. buffer (pH 5) and 4 micro litres of 30% H202
was used as
Asymptomatic, non-microfilaraemic individuals with substrate. The colour developed was read at 415 nm.
no symptoms of the disease were considered endemic
Western blot: Bmmf (S) antigens were separated on
normals (EN). Non-endemic normal sera (NEN) were
10% SDS-PAGE minigel under reducing conditions
collected from Medical students admitted from nonalong
with molecular weight markers. Antigens were
endemic areas such as the states of Himachal Pradesh
transferred to nitrocellulose paper (Hoefer) at 100 mA
and Jammu & Kashmir. Ascaris, leishmania, malaria
for 1 h and blocked with 4% skimmed milk powder for
and hookworm sera from non-endemic areas (a kind
1 h at room temperature (RT). Supernatants from the
gift from Sarika Gupta and Dr. Sushma Rathour,
positive wells as well as ascitic fluid of the clones
Banaras Hindu University) were also screened in order
(1:5000 dilution) were added and incubated at room

32
Monoclonal antibody Bm Ab120 in diagnosis of filariasis
JPD : Vol. 28 (1), 2004
temperature overnight. After washing 5 times with upon high ELISA titres.
PBS/T anti-mouse horseradish peroxidase (1:2000)
Properties of mAb; This monoclonal antibody was of
was added and strips incubated for 1h. This was
the IgM type as shown by isotyping by ELISA.
followed by 5 washes and bands were developed by
Immunoblot revealed that the mAb specifically
adding diaminobenzidine/H2O 2substrate.
reacted with a 120kDa antigen of Bmmf (S) as shown
Isotyping: Ig isotypes produced by hybridomas were in Fig.l. The mAb will henceforth be referred to as Bm
determined by using mAb-based mouse Ig isotyping Ab 120. The ability of this mAb to detect antigens from
kit (Pharmingen).
different stages of the parasite life cycle is shown in
Fig.2. Bm Ab 120 showed high reactivity with Bmmf
Ethical considerations: All animal experiments were
(S) and A(S) antigens but low reactivity with L3 (S)
carried out with prior permission from the Institute
antigens of B.malayi.
Animal Ethics Committee (IAEC). Blood samples
were collected from microfilarial carriers during Screening of endemic population using Bm Ab 120:
routine survey of the Public Health Department, Govt. IgM antibodies purified from ascitic fluid were used as
of Maharashtra, after informed consent.
RESULTS
Selection of monoclonal antibodies: Two out of 96
hybrids showed the presence of antibodies towards the
antigenic determinants present in whole Bmmf (S)
antigen. These two hybrids were cloned further to
obtain several mAbs, of which one was selected based
120 kDa
TABLE : Detection of filarial antigen in different groups
of sera using Bm Ab120 as probe in Sandwich ELISA
Type of sera No. of sera No. positive (%)
Screened FSIgG- Anti-Bmmf(S)-
BmAb120 BmAb120
Mf carriers 50 40(80) 44(88)
Acute 10 8(80) 8(80)
Grade I CP 15 11(73) 10(67)
Grade II CP 15 9(60) 8(53)
Grade III CP 10 5(50) 3(30)
'Occult' 40 14(35) 10(25)
EN 50 9(18) 6(12)
NEN 15 0(0) 0(0)
Mf carriers = Microfilaraemic carriers
CP = Chronic pathology,
EN = Endemic normals
NEN = Non-endemic normals.
Absorbance values of Mean + 2SD of the 50 EN screened
was determined as the cut off value to distinguished between
EN and infected individuals.
97.4 kDa
66 kDa
45 kDa
29 kDa
Fig. 1. Immunoblot showing recognition of 120kDa component of
Bm mf(S) by Bm Ab120.

JPD : Vol. 28 (1), 2004
Monoclonal antibody Bm Ab120 in diagnosis of filariasis
33
OD VALUES AT 490 nm
Absorbance
Absorbance
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2
1.6
1.2
0.8
0.4
0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1000 4000 8000 16,000
RECIPROCAL TITRES OF BmAb 120
Bm mf ES Bm A S Bm L3 S Bm mf S
Fig. 2. Reactivity by ELISA of the different filarial antigens with the
monoclonal antibody BmAb 120 (5 µg/ml protein).
MFC CP OC EN NEN
Sera screened
Fig. 3. Result shown are Mean + SD obtained with sera diluted 1:100.
MF = mf carriers, CP = clinical pathology, OC = suspected occult
filariasis, EN = endemic normals, NEN = non-endemic normals.
MFC CP OC EN NEN
Fig. 4. Results shown are Mean + SD obtained with sera diluted 1:100.
MF = mf carriers, CP = clinical pathology, OC = suspected occult
filariasis, EN = endemic normals, NEN = non-endemic normals.
a probe in a sandwich ELISA using two polyclonal
antibodies [FSIgG and anti-Bmmf (S)] as capture
antibodies. The results are summarized in the
Table. The OD values for circulating filarial antigen
using anti- Bmmf (S) antibodies were observed to be
higher in all groups of sera compared to use of FSIgG
(Figs.3 & 4). Absorbance values of Mean + 2SD of the
50 EN screened was determined as the cut off value to
distinguish between EN and infected individuals. With
FSIgG as capture antibody, the assay was able to detect
40/50 microfilaraemic carriers and 33/50 clinical
cases. Nine of 50 endemic normals also had antigen
levels beyond the cut-off. When anti-Bmmf (S)
antibodies were used to capture filarial antigens, four
more mf carriers were detected while the number of
clinical cases detected was fewer by four. Only 6 EN
were picked up as positive by this assay. Greater
numbers of acute and early chronic cases were picked
up as compared to Grade III stage of disease by both
assays. Among the sera of probable occult filariasis
screened, only 35% and 25% were above the cut-off
titres of which TPE constituted the majority of cases.
None of the fourteen non-endemic normals sera or sera
of other parasitic infections screened were positive by
either combination.
DISCUSSION
Various filarial antigens have been implicated in
protective, pathogenic and allergic reactions in the
host. Identification of specific filarial antigens would
facilitate development of methods for detection of
established infection. Several monoclonal antibodies
have been developed against filarial antigens of which
some are species and stage specific (Burkot et al.,
1996) while others cross-react with epitopes of other
nematodes (Subrahmanyam et al., 1990; Zheng et al.,
1987). Some monoclonal antibodies are of diagnostic
utility (Weil et al., 1987; More & Copeman, 1990)
while some others have been used to confer passive
immunity to the host (Parab et al., 1988). Sandwich
ELISA using polyclonal and monoclonal antibodies
against recombinant filarial antigens have also been
used for screening of filarial sera (Theodore & Kaliraj,
1996). Two commercially available kits employing
monoclonal antibodies directed against animal filarial

34 Monoclonal antibody Bm Ab120 in diagnosis of filariasis JPD : Vol. 28 (1), 2004
parasites that have been widely used in
epidemiological studies in filariasis include the rapid
format test kit developed by ICT Diagnostics
(AMRAD, Australia) and the Trop Bio Test. Various
studies have confirmed the usefulness of these kits in
detecting mf carriers with high sensitivity in different
geographical areas (Bhumiratana et al, 1999;
Simonsen et al, 1999). However, epidemiological
studies have revealed that these tests show poor
sensitivity in detecting amicrofilaraemic individuals
with pathology and adult worm burdens (Nguyen et al,
1999). The clinical manifestations in lymphatic
filariasis range from acute symptoms (lymphadenitis,
fever and chills), chronic presentations (elephantiasis
and hydrocele) and occult manifestation (TPE,
bronchial asthma, arthritis etc.). Most of the above
individuals do not show the presence of mf in
peripheral blood and hence may escape detection by
ICT and Og4C3 assays that show high sensitivity in
detecting microfilarial carriers. It is very difficult to
diagnose atypical filarial manifestations and advise
optimal DEC treatment for clinical relief and cure.
Diagnosis offilaria etiology and morbidity control is
also important in filaria control programme. Our
studies with ICT have revealed 100% sensitivity in
detecting mf carriers while only 1 of 6 clinical filarial
cases was positive for antigen (Alli et al, 2001).
Though ultrasonography techniques are available to
screen amicrofilaraemic individuals carrying adult
worms (Amaral et al, 7994), it is not a viable
alternative to screen large populations. This
necessitates the need to develop specific agents to
detect antigenemia in not only mf carriers but also
across a clinical spectrum of filariasis including those
harbouring occult infections.
Microfilariae in peripheral circulation of the host are
picked by the mosquito vector where they undergo
further development into the infectious L3 stage.
Different circulating antigens from the microfilarial
stage of the parasite have shown diagnostic potential
(Balaji Ganesh et al, 2001; Alli et al, 2000,
Chenthamarakshan et al, 1996). Development of
mAbs against mf antigens could therefore facilitate the
identification of such antigens. Therefore mAbs were
generated using Bmmf (S) extract and the resulting
mAbs used to screen a spectrum of population in an
endemic area.
Bm Ab 120 was of the IgM isotype and specifically
recognized a 120 kDa fraction of B. malayi mf (S)
antigen. The mAb also reacted with Bm mf ES antigen,
BmA antigen and Bmmf ES antigen but poorly with
BmL3 antigen. This is probably because the 120 kDa
antigen is expressed highly in the mammalian stage of
the parasite life cycle and less in the mosquito vector.
Immunofluoresence studies revealed that the antigen
was localized on the surface of the mf and adult stages
of the parasite and not the L3 stage (data not shown).
Circulating filarial antigen recognized by Bm Ab 120,
thus, seems to be present in the mammalian stages of
the filarial parasite life cycle.
BmAbl20 was used as a probe in a sandwich ELISA
using two different polyclonal antibodies, FSIgG and
anti-Bm mf(S) as capture antibodies. FSIgG -Bm Ab
120 could detect specific antigen in 80% mf carriers,
66% of clinical filariasis and also picked up 18% of EN
while anti Bmmf(S)- Bm Ab 120 sandwich ELISA
could detect 88%, 58% and 12% of mf carriers,
symptomatic cases and EN respectively. The increased
sensitivity of the latter in detecting microfilarial
carriers may be due to increased specificity of specific
antibody in anti-Bmmf (S) serum as compared to
FSIgG isolated from filarial patient sera. The
increased number of positives among the endemic
normals with FSIgG resulted in lower difference in
mean OD values between filarial and endemic normal
groups as compared to the anti-Bmmf (S)- Bm Ab 120
assay where mean OD of mf carriers was 3 times more
than the endemic normals screened. The 80-88%
sensitivity of the assays in detecting mf carriers in this
study was lower than those reported earlier for the ICT
or Trop Bio tests (90- 100%). However, a significantly
high number of clinical cases were detected by this
assay. The fact that many of the clinical cases, even
though absent for microfilaraemia, showed presence
of antigen was probably indicates the presence of
active infecton. The assays could also detect filarial
specific antigen in 25-35% of 'occult' cases, most of
them TPE cases and 2 cases of bronchial asthma,

JPD : Vol. 28 (1), 2004
Monoclonal antibody Bm Ab120 in diagnosis of filariasis
35
suggesting filarial etiology further emphasizing the Clin. Biochem.: 16 (2),207-10
need to rule out filariasis in individuals with such Alli R, Bhandari YP, Reddy MVR and Harinath BC. 2000.
atypical manifestations, particularly in children. Seroreactivity of purified Brugia malayi microfilarial soluble
and excretory-secretory antigens in different clinical
Antigen was not detected in pooled sera from few
presentation of bancroftian filariasis. Ind. J. Exp. Biol, 38, 791-
patients from non-endemic areas suffering from
96.
infections with hookworm, ascaris, malaria and
Amaral F, Dreyer G, Figueredo-Silva J, Noroes J, Cavalcanti A,
leishmania showing the monoclonal antibody is Samico SC, Santos A and Coutinho A. 1994. Live adult worms
specific to filarial antigen. Screening of malayan sera detected by ultrasonography in human Bancroftian filariasis.
could also reveal whether Bm Ab 120 is useful in Am J Trop Med Hyg.: 50 (6),753-7
detecting B.malayi infected individuals as the mAb Balaji Ganesh B, Kader AM, Agarwal GS, Reddy MVR and
reacts with both B.malayi mf and adult worms. Twelve Harinath BC. 2001. A simple and inexpensive dot-blot assay
and eighteen percent of the endemic normals screened using a 66-kDa Brugia malayi microfilarial protein antigen, for
diagnosis of bancroftian filarial infection in an endemic area,
were positive by the assays using different polyclonal
Trans. Roy. Soc. Trop. Med. Hyg. 95, 168-169.
antibodies. This is in agreement with some of the
Bhumiratana A, Koyadun S, Suvannadabba S, Kamjanopas K,
studies using ICT that have shown 14%- 24.7% of
Rojanapremsuk J, Buddhirakkul P and Tantiwattanasup W.
endemic normals as antigenemia positive 1999. Field trial of the ICT filariasis for diagnosis of
(Bhumiratana et al, 1999. Njenga et al, 2001). Many Wuchereria bancrofti infections in an endemic population of
endemic 'normals' are believed to harbour pre-patent Thailand. Southeast Asian J Trop Med Public Health : 30
or cryptic infections or have undetectable levels of
(3),562-8.
microfilaraemia but show no overt clinical symptoms Braga C, Dourado MI, Ximenes RA, Alves L, Brayner F, Rocha A
and Alexander N. 2003. Field evaluation of the whole blood
resulting in considerable background positives. We
immunochromatographic test for rapidbancroftian filariasis
conclude that Bm Ab 120, when used with other diagnosis in the northeast of Brazil. Epub May-Jun; 45 (3), 125-
suitable polyclonal antibodies may help in the 9.
detection of circulating filarial antigen in Burkot T R, Kwan -Lim GE & Maizels RM. 1996. A novel 95-
identification of infected individuals living in an kilodalton antigen of Wuchereria bancrofti infective larvae
endemic area. Further multicentric studies are identified by species-specific monoclonal antibodies, Infect.
required for strengthening the evidence base and for
Immun. 64 (2), 485-488.
deciding the possible practical implication in Chandrasena TG, Premaratna R, Abeyewickrema W and de Silva
NR. 2002. Evaluation of the ICT whole-blood antigen card test
individual and community diagnosis of filariasis.
to detect infection due to Wuchereria bancrofti in Sri Lanka.
ACKNOWLEDGEMENTS
Trans R Soc Trop Med Hyg : 96 (1), 60-3.
Chenthamarakshan V, Reddy MVR and Harinath BC. 1996.
This work was supported by the Department of
Diagnostic potential of fractionated Brugia malayi
Biotechnology, New Delhi. The authors are grateful to microfilarial excretory /secretory antigen for bancroftian
Shri. Dhirubhai Mehta , President of Kasturba Health filariasis. Trans. Roy. Soc. Trop. Med. Hyg. 90 : 252-254
Society, Dr. P. Narang, Dean, MGIMS and Dr. G.C. Eigege A, Richards FO Jr, Blaney DD, Miri ES, Gontor I, Ogah G,
Mishra, Director, NCCS for their keen interest and Umaru J, Jinadu MY, Mathai W, Amadiegwu S and Hopkins
encouragement in this work.
DR. 2003. Rapid assessment for lymphatic filariasis in central
Nigeria: a comparison of the immunochromatographic card test
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Journal of Parasitic Diseases
Vol.28 (1) June 2004, pp. 37-44
Anthelmintic efficacy of extract of Stephania glabra and
aerial root extract of Trichosanthes multiloba in vitro : two
indigenous plants in Shillong, India
1
V TANDON*, LM LYNDEM, PK KAR, P PAL, B DAS, HSP RAO
Department of Zoology, North Eastern Hill University, Shillong-793022, India
1
Department of Chemistry , Pondicherry University, Pondicherry, India.
In the traditional medicinal practices in Meghalaya (Northeast India) aqueous concoction of the powders of
the dried rhizome of Stephania glabra and aerial root of Trichosanthes multiloba is used as anthelmintic
against intestinal worms. To ascertain their efficacy, the alcoholic crude extracts of these materials were
tested against the various helminth parasites - nematode: Heterakis gallinarum, Ascaridia galli, Ancylostoma
ceylanicum and Ascaris suum; cestode: Raillietina echinobothrida and trematode: Fasciolopsis buski in
dosages ranging between 25-100 mg/ml in 0.9% phosphate buffered saline (PBS, pH 7.2) at 38 ± 1°C. The
controls, kept in PBS, survived for average 22 h (trematode), 72 h (cestode) and 55->380 h (nematodes).
Treatment with S. glabra showed pronounced effect on the cestode and trematode; a dose-dependent
gradual decline in physical motility was observed in R. echinobothrida and F. buski. The isolated active
principle (Compound X) of the rhizome pulp also showed good efficacy against F. buski. In contrast, much
less effect was revealed in respect of nematodes; in treatments with 50 mg/ml all the smaller nematodes
attained paralysis within 7-10 h, whereas A. suum remained unparalyzed even after 40 h. In treatments with
Trichosanthes alone the onset of paralysis in all the test worms took much longer than Stephania, though in
concoction of the two crude extracts the paralytic effect was discernible in less than half the time taken for
individual treatments. The phytochemical(s) of S. glabra seems to be effective against platyhelminth
parasites that have a tegumental interface, but not so against cuticle-covered nematodes.
Keywords: Anthelmintic; Cestode; Nematode; Parasites; Stephania glabra; Trichosanthes multiloba;
Trematode
A
large number of plants with medicinal
properties have been reported from India
(Chopra et al., 1956; Dhar et al., 1965, 1968, 1973;
Dhawan et al., 1980; Rao and Krishnaiah, 1982;
Bhakuni et al., 1988; Shilaskar and Parasher, 1989)
and some of them have been documented as curative
against worm infections (Perry, 1980; Dharma, 1985;
Chhabra et al., 1990). Meghalaya, one of the
biodiversity hotspots of Northeast India, has a wide
variety of plants used by the natives as curatives
against worm infections (Rao, 1981). Besides
* Corresponding Author
Flemingia vestita (family Fabaceae) that is used
widely as an anthelmintic against gastrointestinal
worms (Tandon et al., 1997), two other plants,
Stephania glabra Miers (family Menispermaceae) and
Trichosanthes multiloba Clarke (family
Cucurbitaceae), are similarly used in the traditional
medicinal system. While the rhizome pulp of S. glabra
is used as anthelmintic locally, the aerial root of T.
multiloba is used in concoction with S. glabra pulp for
more effective cure.
With a view to establishing anthelmintic efficacy of
the above-mentioned plants and to find out clues to the
plausible mode of their action, a thorough study

38 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004
seemed desirable. The present study, therefore, aimed using about 300 ml of mixed solvent at each ratio.
at investigating the efficacy of the phytochemicals on
From the rhizome pulp and peel only one major
the model nematode, cestode and trematode parasites
isoflavone could be isolated, the chemical constitution
of zoonotic significance. Physical motility and of which is yet to be ascertained; hereafter this is
survival time including the onset of the paralytic state
referred to as Compound X. For T. multiloba, due to
and alterations in the surface architecture of the
very less quantity of the crude extract recovered from
parasites constitute the parameters of activity.
the available material, isolation of active components
MATERIALS AND METHODS
could not be pursued.
Plant crude extract: Fresh rhizomes of S. glabra and Parasites: Parasites (nematodes: Heterakis
aerial roots of T. multiloha were collected in winter gallinarum and Ascaridia galli from domestic fowl
from Mawsynram, a small hamlet, about 60 km from and Ascaris suum from pigs; cestode: Raillietina
Shillong (Meghalaya). After washing thoroughly with echinobothrida from domestic fowl; trematode:
water the rhizomes were thinly peeled and the pulp was Fasciolopsis buski from pigs) were collected in
diced into small pieces; the aerial roots were also phosphate buffered saline (PBS; pH 7.2) from freshly
chopped into small unpeeled pieces. Each of these slaughtered hosts at the local abattoirs. Ancylostoma
materials was soaked separately in rectified alcohol ceylanicum was obtained from infected hamsters
for about a month; in the case of S. glabra, the maintained in the laboratory. The parasites were
alcoholic contents turned dark brown, whereas the exposed to different treatments within 1 h of
solvent with T. multiloba aerial roots attained only a collection.
golden tinge. The alcoholic solutions thus obtained
Treatments: The test parasites were incubated at 38 ±
were distilled in a reflux condenser for isolating the
1°C in 0.9% phosphate buffered saline (PBS, pH 7.2)
concentrated crude extract.
containing crude extract in concentrations of 25, 50
Finally 200 g dry crude extract was obtained from 4 kg and 100 mg/ml supplemented with 1 % DMSO. While
pulp, and 40 g from 2.5 kg peel of S. glabra. From F. buski and A. suum were kept singly for each test, H.
about 5 kg aerial roots of T. multioloba, 50 g crude gallinarum and A. ceylanicum were incubated in
extract was retrieved.
batches of 5 worms, each of approximately same size.
For R. echeinobothrida, on the other hand, 5 strobila,
Compound X: Purified active component from the
each with scolex and of approximately equal length,
crude extract of S. glabra rhizome pulp and peel was
were used. For each experiment three replicates were
isolated following the method of Rao and Reddy
used. The reference drugs praziquantel (PZQ,
(1991). The alcoholic crude extract of the plant
cestocide), oxyclozanide (flukicide) and mebendazole
material was mixed well with hexane and the
(nematocide) were used in various concentrations with
supernatant solution was decanted. This process was
1 % DMSO, as established in earlier studies (Visen et
repeated 20 times. The extract was reduced in volume
al., 1987; Roy and Tandon, 1996; Tandon et al., 1997).
through distillation and passed through silica gel
Controls were maintained in 0.9 % PBS with 1%
column using hexane, benzene and ethyl acetate as
DMSO at 38 ± 1°C. The parasites, both in control and
solvents. The column was prepared using hexane and
treated, were brought back to the slightly warm PBS to
benzene mixture (6:4) and silica gel 100-200 mesh
examine their motility at every 15 min gap and the time
(about 350 g). The sample (10 g) was mixed with silica
taken for the reversible loss of motility (paralysis) and
gel 60-120 mesh and loaded on top of the column.
death (no motility) was recorded as per the procedure
Elution was carried out with hexane: benzene mixture
followed in earlier similar study (Tandon et al., 1997).
successively at the ratio of 6:4, 5:5, 4:6 and so forth to
pure benzene and thereafter with benzene: ethyl Scanning electron microscopy: Soon after they
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
Anthelmintic efficacy of rhizome extract
39
echeinobothrida, F. buski and A. ceylanicum), treated high efficacy at a concentration of 1.0 mg/ml, with
with various dosages of the rhizome-pulp extract of S. paralysis ensuing in less than 1 h.
glabra, were fixed along with their respective controls
The aerial root extract of T. multiloba showed a
in 10% neutral buffered formalin at 4°C for 24 h,
similar pattern of efficacy; at the concentration of
washed in PBS and dehydrated with ascending grades
l00 mg/ml, paralysis occurred at 2.19 ± 0.001 h,
of acetone to pure dried acetone. The specimens were
2.41 ± 0.52 h, 5.26 ± 0.072 h and 11.02 ± 0.003 h in
then critical-point dried using liquid carbon dioxide as
the case of R. echinobothrida, F. buski, A.
the transitional fluid or treated with tetramethylsilane
ceylanicum and A. galli, respectively. This efficacy
following Roy and Tandon (1991). The gold-coated
is more or less comparable to that of S. glabra.
specimens were observed using LEO 435 VP SEM or
However, in the concoction of the two plant
JEOL at electron-accelerating voltages ranging materials (1:1) the time taken for paralysis in the
between 10-20 kV.
test parasites lowered significantly in comparison
Statistical analysis: Data were statistically analyzed to the treatment with the crude-pulp extract of S.
and presented as mean ± SEM (n = 3). Comparisons of glabra alone, indicating a synergistic effect of the
the paired mean values between the experimental and two at a dosage of 50 or 100 mg/ml.
respective reference were calculated using Student's
All the nematode species showed a negligible effect as
t-test (Croxton et al., 1982) and differences with
they survived the various treatments for comparatively
P

40 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004
Figs. 1-6. Surface architecture of the parasites following treatment with
S. glabra crude rhizome pulp extract (100 mg/ml):
1, 2. Fasciolopsis buski- ventral tegument
1. Control, showing the spination of the normal surface.
(Scale bar = 10µm).
2. Treated fluke; distortion of the spines, sloughing and
pits are noticeable.
(Scale bar = 30µm).
3, 4. Ancylostoma ceylanicum- cuticle, mid body
3. Control, showing striated surface pattern.
(Scale bar = 2.5µm).
4. Treated; not much distortion of the cuticular
architecture is discernible even at higher
magnification.
(Scale bar = 5µm).
5, 6. Raillietina echinobothrida- tegumental surface of the
gravid proglottid
5. Control, showing normal surface contour.
(Scale bar =100 µm).
6. Treated worm; cracks and wrinkles in the tegumental
surface, with clumpy microtriches are evident.
(Scale bar =100 µm).

JPD : Vol. 28 (1), 2004
Anthelmintic efficacy of rhizome extract
41
Table : Efficacy of the rhizome pulp and peel extracts of Stephania glabra and aerial root extract of Trichosanthes
multiloba against various helminth parasites*
Time (h) taken for complete loss of movement
Treatment Raillietina Fasciolopsis Heterakis Ancylostoma Ascaridia Ascaris
(mg/ml) echinobothrida buski gallinarum ceylanicum galli suum
S. glabra
Crude rhizome pulp extract
25 7.4+0.05 5.04+0.02 - 11.38+0.12 - -
a a b
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
a a b a b x
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
Crude rhizome peel extract
25 - - - 11.01+0.02 - -
b
50 5.05+0.13 - - 6.80+0.07 - -
b
a
100 3.01+0.05 - - 3.18+0.04 - -
Compound X
0.5 - 1.37+0.01 - - - -
b
1.0 - 0.74+0.02 - - - -
T. multiloba
Crude aerial root extract
25 3.76+0.01 - - 12.6+0.1 15.58+0.01 -
x b x
50 3.39+0.02 4.07+0.71 - 8.1+0.02 13.7+0.01 -
b b a b
100 2.19+0.01 2.41+0.52 - 5.26+0.07 11.02+0.01 -
S. glabra (pulp) : T. multiloba (1:1)
50 - 1.18+0.03 - 3.21+0.04 - -
a
b
100 - 0.32+0.05 - 2.71+0.04 - -
Praziquantel
0.001 2.9+0.05 - - - - -
a
0.005 0.89+0.04 - - - - -
b
0.01 0.47+0.07 - - - - -
Oxyclozanide
5 - 1.97+0.01 - - - -
b
10 - 1.12+0.02 - - - -
a
20 - 0.68+0.03 - - - -
Mebendazole
2.5 - - - 5.08+0.03 - -
b
5 - - - 3.78+0.02 - -
a
10 - - - 2.51+0.02 - -
* The parasites survived in control (0.1% DMSO in PBS, pH 7.2) for considerably longer periods than in any
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
h; A. galli, 168+0.08 h and A. suum, 384+0.1 h. Values are expressed as mean + S.E.M. (n=3).
- not tested.
a, b
: p-value significant at < 0.01 and < 0.05 level, respectively in comparison to lower dosages.
x
: Not significant.

42 Anthelmintic efficacy of rhizome extract JPD : Vol. 28 (1), 2004
DISCUSSION
treatment with PZQ, are well established
(Mehlhorn et al., 1983; Schmahl and Mehlhorn,
The present study demonstrates that the test plant
1985). Tegumental bubbles of different sizes
materials have a deleterious effect on R.
appeared on the surface of Opisthorchis viverrini
echinobothrida and F. buski. In ultrastructural studies,
treated with PZQ in vitro and in vivo. Thus these
disorientation of the microtriches, spines and scales
ultrastructural changes may represent a generalized
was observed along with severe distortion and
response of the tegumental surface to an obnoxious
deformity of the tegument, breakage and sloughing off
agent (Sirisinha et al., 1984). The same fluke when
of the tegumental surface structures. Cestodes and
treated with amoscanate exhibited severe swelling
trematodes are soft-bodied parasites and the tegument
and pit formation, leading to total disruption of the
is their only or major interface, through which various
surface tegument and suggested that the drug may
basic physiological functions such as digestion and
have caused an imbalance in osmosis, resulting in
absorption take place. The plant-derived components
impaired ion transfer (Sobhon et al., 1986). The
seem to affect this normal physiological functioning
changes in the tegumental architecture on treatment
causing paralysis and subsequent mortality of the
with various test materials of plant origin suggest
parasite, as has been described before in case of R.
that the plant products bring about permeability
echinbothrida and F. buski on being treated with F.
changes in the tegument of the worm.
vestita and its active component genistein (Roy and
Tandon, 1996; Tandon et al., 1997; Pal and Tandon, Disruptions of the cuticular interface and/or
1998). intestinal epithelium and degenerative changes
even in the subcuticular region have been reported
Tegumental distortion and severe vacuolization on
in several nematode species exposed to
exposure to flukicidal drugs have been observed in
anthelmintics in vitro (An, 1990; Storte et al., 1990;
several species of trematodes (Jiang and Xia, 1992;
Mackenstedt et al., 1993; Rothwell and Sangster,
Schmahl, 1993; Stitt and Fairweather, 1993; Xu-
1996). Though there was also an orderly decrease in
Lin et al., 1994; Anderson, and Fairweather, 1995);
the time taken for paralysis with increasing
the extent of damage induced was reported to
concentration of the plant-derived components in
increase with the exposure time. Similar changes
the nematode species in the present study, the onset
were noticed in case of cestode parasites (Delabreof
paralysis in them took much longer time than the
Defayolle et al., 1989; Perez et al., 1994; Pal and
platyhelminth parasites. The synergistic effect of S.
Tandon, 1998). In Fasciola hepatica, treatment
glabra and T. multiloba concoction (1:1) at 50 or
with diamphenetide caused blebbing of the 100 mg/ml showed more efficacious results
tegument and the blebs increased in size with an
compared to the effect in treatments with individual
increase in the dosage; surface pitting was also
plant materials.
observed and the mid body region of the tegument
was stripped off to expose the basal lamina beneath It can be hypothesized that the paralysis of the flat-
(Fairweather et al., 1987). The ultrastructural bodied helminth parasites on exposure to the plantchanges
in the tegument are linked to a possible derived components is attributable to the severe
mode of action of the drug as an inhibitor of protein distortion, degeneration and disruption of the
synthesis (Anderson and Fairweather, 1995). tegumental architecture of the parasite. The plant-
Vacuolization and contraction in the parasite body derived components do not seem to be much
2+
surface are closely related to the levels of Ca effective against the cuticle-covered nematodes. It
concentration of the media used (Bricker et al., may further be assumed that these phytochemicals
1982; Xiao et al., 1984). Disturbances in ion flux have a vermifugal action in that the paralyzed
across the membrane, leading to changes in the parasites are removed from the host by peristaltic
tegumental integrity in different trematodes on movements of the intestine.

JPD : Vol. 28 (1), 2004
Anthelmintic efficacy of rhizome extract
43
ACKNOWLEDGEMENTS
Carica papaya seeds. Indian J. Pharmacol. 27:335-336.
Dharma AP. 1985. Tanaman obat traditional Indonesia, Jakarta,
This work was supported by GBPIHED (Ministry of
PN. Balai Pusterka.
Environment & Forests, GOI)-sponsored project to
Dhawan BN, Dubey MP, Mehrotra BN, Rastogi RP and Tandon JS.
VT and DRS-III project of the University Grants
1980. Screening of Indian plants for biological activity: Part IX.
Commission, GOI, in the Department of Zoology, Indian J. Exp. Biol. 118(6): 594-606.
North Eastern Hill University, Shillong. Thanks are
Fairweather I, Anderson HR and Baldwin TM. 1987. Fasciola
also due to the Head, Department of Chemistry, hepatica. tegumental surface alterations following treatment in
Pondicherry University, for allowing the usage of vitro with deacetylated (amine) metabolite of diamphencthide.
laboratory facility for extraction of active components Parasitol. Res. 73(2):99-106.
of plant materials and the Head, Anatomy Department, Jiang JX and Xia DG. 1992. Transmission electron microscopical
A.I.I.M.S., New Delhi and Director, R.S.I.C., NEHU, observation on the effects of praziquantel and albendazole on
Paragonimus heterotremus in rats. Chin. J. Parasitic Dis. Contr.
Shillong for SEM facility.
5(4):264-266.
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Paris J and Petavy AE. 1989. Echinococcus multilocularis Rao VS and Krishnaiah KS. 1982. Note on the comparative
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levopraziquantel. Chin. Med. J. 107(10):771-774.

Journal of Parasitic Diseases
Vol. 28 (1) June 2004, pp. 45-56
Anthelmintic efficacy of genistein, the active principle of
Flemingia vestita (Fabaceae): Alterations in the activity of the
enzymes associated with the tegumental and gastrodemal
interfaces of the trematode, Fasciolopsis buski
PRADIP KUMAR KAR and VEENA TANDON*
Department of Zoology, North-Eastern Hill University, Shillong - 793022
Acid phosphatase (AcPase) and alkaline phosphatase (AlkPase) were found widely distributed in
tegumental tissues, i.e., tegument, subtegument and somatic musculature as well as in the gastrodermis of
the intestinal fluke Fasciolopsis buski. Adenosine triphosphatase (ATPase) and 5'-Nucleotidase (5'-Nu), to
the contrary, were localized in the tegumental tissues only while protease was associated with the lattermentioned
tissues. After exposure to the various test materials (crude root-peel extract of Flemingia vestita
20 mg/ml, its active component genistein 0.5 mg/ml and the reference drug oxyclozanide 20 mg/ml
concentration) a significant decline in the visible stain intensity of all these enzymes except protease was
observed in the aforementioned regions. Quantitatively, the activities of AcPase, AlkPase, ATPase, and 5'-
Nu were found to diminish by 39-42.75%, 37.5-44.23%, 32.9-54.5% and 41.9-51.9%, respectively after
treatment. The reference drug showed somewhat similar effect like the crude extract of F. vestita.
Keywords: Acid phosphatase, Adenosine triphosphatase, Alkaline phosphatase, Anthelmintic, Flemingia
vestita, Genistein, 5'-Nucleotidase, Phytochemicals, Protease, Tegumental enzymes, Trematode
he presence of several enzymes viz., acid metacercarial cyst of Bolbogonotylus corkumi and
Tphosphatase (AcPase), alkaline phosphatase adult Fibricola seoulensis, an intestinal trematode of
(AlkPase), adenosine triphosphatase (ATPase) and 5'- human and rodents, showed moderate AcPase activity
Nucleotidase (5'-Nu) has been detected both (Walker and Wittrock, 1992; Huh, 1993). The
histochemically and biochemically in a number of functional characteristics of the tegument and
helminth parasites, wherein these enzymes have been intestinal wall were studied in mature Fasciola
found to be closely associated with the tegument or hepatica after treatment with luxabendazole, and
cuticle, subtegument, somatic musculature and gut alterations were noted in the activities of AcPase,
(Kwak and Kim, 1996; Buchmann, 1998; Fetterer and ATPase, inosine triphosphatase and succinate
Rhoads, 2000). AcPase and AlkPase occur widely in dehydrogenase (Gorchilova et.al., 1990). The aqueous
the tegument of trematodes, cestodes and extract of some medicinal plants viz., Butea
acanthocephalans. In adult cestodes AlkPase is usually monosperma, Embelia ribes and Roltlesia tinctoria
the most active, whilst AcPase tends to predominate in caused reduction in both AcPase and AlkPase
the trematode tegument (Barrett, 1981). The activities of Paramphistomum cervi in vitro (Chopra et
al., 1991). Praziquantel (PZQ), on the other hand,
* Corresponding Author
induced AlkPase activity on the surface of the adult
Schistosoma mansoni (Fallon et. al., 1994).

46 Anthelmintic efficacy of genistein
JPD : Vol. 28 (1), 2004
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
involved in digestion and/or absorptive function in liver/intestinal/blood/lung flukes, haematophagous
cestodes (Poljakova et al., 1983). McCracken and and tissue dwelling nematodes, and cestodes (review-
Taylor (1983) reported biochemical effects of Trap and Boireau, 2000). In view of the differences in
thiabendazole and cambendazole on Hymenolepis the inhibition properties from their host
diminuta in vivo; the failure of glucose uptake could counterparts and specific capacities for
lead to the depletion of parasite AlkPase activity. immunomodulation the parasite proteases are
The AcPase activity in the isolated brush border currently being proposed as major immuno- and
membrane of H. diminuta was inhibited chemotherapeutic targets (Hawthorne et al., 2000;
particularly by levamisole, 2-mercaptoethanol and Hartmann and Lucius, 2003) and their potential in
EDTA (Pappas, 1988).
the development of vaccines is being exploited
The broad spectrum anthelmintics parbendazole and
(Dalton et al., 2003; Williams et al., 2003).
piperazine adipate showed a marked reduction in the In a previous study, genistein, the active principle of
activity of AcPase activity in the nematodes Ascaridia the root-peel of Flemingia vestita, a leguminous plant
galli and Heterakis gallinae but the activities of lactate with edible tuberous roots, was shown to induce
dehydrogenase and AlkPase were not affected alterations in the activity of tegumental enzymes in
significantly (Sharma et al., 1987). Some drugs like cestodes (Pal and Tandon, 1998). In view of the
phenothiazine, diethylcarbamazine and centperazine putative transtegumental functioning of this
altered the metabolism and inhibited the formation of phytochemical, we extended the study to trematode
mitochondrial energy, thus depriving the parasite worms in order to ascertain if the plant-derived
Setaria cervi of ATP (Agarwal et al., 1990). components influence the activity of enzymes
Tetramisole and levamisole also caused inhibition of associated with the tegumental and gastrodermal
++
the specific activities of AcPase and Mg -dependent interfaces of these parasites.
ATPase in A. galli, though AlkPase activity increased
MATERIALS AND METHODS
in the presence of both these drugs (Aggarwal et al.,
1992). Thiabendazole and mebendazole changed the
Parasites and test materials; Mature Fasciolopsis
activity of AcPase in Nippostrongylus brasiliensis in
buski were collected from the intestine of swine (Sus
infected mice (Mishra and Srivastava, 1990).
scrofa) obtained from the local abattoirs in 0.9%
Rafoxamide also affected the key dehydrogenases
Phosphate Buffered Saline (PBS, pH 7.2). The crude
involved in the carbohydrate metabolism of Trichuris
root-peel extract of F. vestita was prepared as
globulosa by blocking the glycolytic pathway
described earlier (Tandon et al., 1997). The parasites
depriving the parasite of energy (Parveen et.al., 1992).
were incubated with the crude extract (20 mg/ml),
genistein (0.5 mg/ml) and oxyclozanide, the reference
Proteases are known to play an important role in the
drug (20 mg/ml) concentration (all prepared in 0.9%
pathogenesis of several parasitic infections; besides
PBS with 1% DMSO). The aforementioned
general catabolic functions, these proteolytic enzymes
concentrations were chosen since at these dosages the
may be significant in parasite immunoevasion, paralytic effect of the worm was attained within a
excystment/evagination/encystment, and cell and shorter time frame as compared to lower
tissue invasion (Park et al., 2001; Sajid and Mckerrow,
concentrations (Roy and Tandon, 1996). Three
2002). Proteases represent a very heterogeneous group
replicates for each set of incubation medium were
of proteins among parasites (Wijffels et al., 1994) and
used. On attaining the paralytic state after
have been isolated and characterized from the treatment the trematodes were further processed for
excretory/secretory products or tissue extracts of
histochemical and biochemical studies, along with

JPD : Vol. 28 (1), 2004
Anthelmintic efficacy of genistein
47
containing moist filter paper. At 5 min intervals the
sections were inspected to make sure that they were not
too dry, in which case further buffer must be added.
After incubation the preparation is dried without
washing and subsequently dehydrated in alcohol,
cleared in xylene and mounted in a synthetic medium.]
The protease activity is shown as a clear area where the
gelatin film and its contained granules have
disappeared. Histological, but not intracellular,
definition is obtained.
Biochemical assays:
AcPase and AlkPase : AcPase and AlkPase activities
were assayed by estimating the p-nitrophenol product
following the method as given by Plummer (1988)
with necessary modification in the concentration of
the buffer and substrate. One unit of AcPase or AlkPase
activity is defined as that amount which catalyzed the
formation of 1 mM of p-nitrophenol/h at 37°C.
ATPase: ATPase activity was assayed by estimating
the free phosphate released, following the method of
Kaplan (1957) with Na-ATP as the substrate. One unit
of ATPase is defined as the amount which catalyzed the
release of 1 µmole of phosphate / h at 37°C from ATP.
5'-Nu: The enzyme activity was assayed by estimating
the free phosphate released following the method of
Bunitian (1970) using AMP as the substrate. One unit
of 5'-Nu activity is defined as that amount which
catalyzed the release of l µmole of phosphate/h at 37°C
from AMP.
Protein: The protein content was estimated following
the method of Lowry et al. (1951) using bovine serum
albumin as a standard.
one set of control specimens (maintained in 1%
DMSO in PBS).
Histochemical localization:
AcPase: The AcPase activity was demonstrated
following the modified lead nitrate method of
Takeuchi and Tanoue as described by Pearse (1968),
wherein β-glycerophosphate was used as the substrate.
A brownish precipitate indicates sites of AcPase.
AlkPase: A modified coupling azo-dye method
described by Pearse (1968) was used for the
determination of AlkPase activity using Fast violet-B.
The sites of AlkPase activity are coloured brown with
salts, nuclei dark blue.
ATPase: For the demonstration of ATPase activity,
calcium method using Na-ATP as the substrate, after
Maengwyn-Davies et al., as described by Pearse
(1968) was followed. The activity of ATPase shows as
a blackish brown deposit.
5'-Nu: For the study of 5'-Nu activity the lead method
after Wachstein and Meisel (1957) was employed
using adenosine monophosphate (AMP) as the
substrate. Yellow deposits of lead sulphide indicate
sites of 5'-Nu enzyme activity.
Protease: For the demonstration of protease the gelatin
method of Adams and Tuqan (1961) as described by
Pearse (1968) was followed.
[The specimens were fixed in cold (4°C) 4%
formaldehyde-saline for 24 h. They were washed
briefly in water and 10-15 µm cryostat sections were
cut. Sections were mounted on to a blackened
photographic plate, cut to a suitable size. The plate was
prepared by exposing rapid panchromatic quarter-
plate to daylight for 10-15 min, developed in the usual
way and hardened in a thiosulphate-metabisulphite
bath before drying. After mounting, the plates were
allowed to become dry and then the sections and the
surrounding gelatin were lightly dampened with 0.15
M phosphate buffer (pH 7.6). The sections were then
incubated for 30-60 min at 37°C in a petri dish
All the chemicals used in the present study were
procured from Sigma Chemicals, USA or SRL, India.
RESULTS
Controls: Intense AcPase and AlkPase activities were
observed in the tegument and gastrodermis. Other
regions viz., the subtegument and somatic
musculature displayed staining intensity lesser than

48 Anthelmintic efficacy of genistein
JPD : Vol. 28 (1), 2004
Table I: Activities of AcPase, Alkpase, ATPase and 5'Nu in various structures of Fasciolopsis buski in vitro: histochemical localization
Treatment
AcPase AlkPase ATPase 5'-Nu
(mg/ml) T ST SM IN T ST SM In T ST SM IN T ST SM IN
Control
(0.9% PBS)
++++ +++ +++ ++++ ++++ ++ +++ ++++ +++ ++ ++ ++ +++ +++ +++ +++
F. vestita ++ + + ++ + - + ++ - - - - - - + -
crude root peel
extract (20)
Genistein (0.5) + + ++ ++ + - ++ + - - + - - - + -
Oxyclozanide
++ + ++ + ++ - ++ + - - - - - - - -
T- tegument; ST - Subtegument; SM- somatic musculature; IN - intenstine
++++ Very strong activity, +++ Strong activity, ++ Medium activity, + less activity, - No activity

JPD : Vol. 28 (1), 2004
Anthelmintic efficacy of genistein
49
Table II: Biochemical effects of the various plant-derived test materials on F. buski
a b
Treatment Enzyme activity (total /specific ) %decrease*
(mg/ml) AcPase AlkPase ATPase 5'-Nu AcPase AlkPase ATPase 5'-Nu
Control 11.3 ±0.26/ 29.7 ± 3.34/ 509.35 ± 85.9/ 43.3 ± 2.37
(0.9% PBS) 1.48 ± 0.03 3.9 ± 0.44 66.67 ± 11.25 5.67 ± 0.31
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
F. vestita (20) 0.98 ± 0.04 2.47 ± 0.25 46.22 ± 6.72 3.41 ± 0.3
Genistein 6.54 ± 0.26/ 17.3 ± 2.44/ 213.9 ± 18.15/ 20.6 ± 1.87/ 42 42.31 54.5 51.9
(0.5) 1.47 ± 0.06 3.9 ± 0.54 48.06 ± .08 4.6 ± 0.42
Oxyclozanide 6.6 ± 0.25/ 17.46 ± 2.8/ 308 ± 27.3/ 24.84 ± 2.05/ 41.6 42.21 36.3 42.8
(20) 1.2 ± 0.05 3.18 ± 0.51 56 ± 5 4.52 ± 0.4
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)
One unit of enzyme activity is defined as that amount of enzyme which consumes 1.0 µmol substrate/g wet wt tissue/h
a
Specific activity expressed as unit/mg protein
b
* P > 0.05

50 Anthelmintic efficacy of genistein
JPD : Vol. 28 (1), 2004
the tegument (Table I; Figs. 1, 2, 4). Not much of
activity was observed in the general parenchyma or the
structures related to the excretory or reproductive
systems.
The ATPase activity was observed mainly in the
tegument of the fluke. In the subtegument, somatic
musculature and intestinal caeca moderate staining
was demonstrated. Among the structures that were
studied tegument, subtegument, somatic musculature
and gastrodermis showed an equally strong activity of
the 5'-Nu enzyme. The stain intensity was much less in
the muscle layer (Figs. 7, 8, 10,11).
Quantitatively the control parasites showed more
enzyme activities than the treated ones with respect to
all the four enzymes that were tested.
Treatment: The activities of AcPase, AlkPase,
ATPase and 5'Nu in the tegument and gastrodermis
were inhibited after treatment with the plant test
3
Fig. 3: AcPase activity in the cryostat transverse sections: Reduced
enzyme activity in the dorsal and ventral tegument, subtegument,
somatic musculature and intestinal caecum after treatment with the
root tuber peel extract of F. vestita (48 x).
Fig. 1: AcPase activity in the cryostat transverse sections: Control -
Intense enzyme activity in the dorsal tegument (DT), subtegument
(ST) and somatic musculature (SM) (120x).
1
Fig. 2: AcPase activity in the cryostat transverse sections: Controlstrong
enzyme activity in the intestinal caecum (IN) (120x)
2
4
Fig. 4: AlkPase activity in the cryostat transverse sections: Intense
enzyme activity in the dorsal tegument, subtegument, somatic
musculature and gastrodermal lining of control flukes (48x).

JPD : Vol. 28 (1), 2004
Anthelmintic efficacy of genistein
51
Figs. 5-6: AlkPase activity in the cryostat transverse sections
Fig 5: Enzyme activity in the intestinal caecum after treatment with
the active component of F. vestita, genistein (120x).
5
Fig. 9: 5'-Nu activity in the cryostat transverse sections of F. buski,
Mild enzyme activity detectable in sections after treatment with root
tuber peel extract of F. vestita (48x).
9
Figs 10-11: ATPase activity in the transverse cryostat sections.
6
Fig 6: Reduced enzyme activity in the ventral tegument,
subtegument and somatic musculature after genistein treatment
(48x).
Figs. 7-8: 5'-Nu activity in the cryostat transverse sections of F.
buski
10
Fig. 10: Control- enzyme activity demonstrated in the dorsal
tegument along with somatic musculature (48x).
Fig.7: Control- section showing
intense enzymatic activity in the
tegument, subtegument and somatic
musculature (48x)
7
8
Fig. 8: Controlsections
showing
enzyme activity in the
intestinal caecum and
(48x).
11
Fig. 11: Control- enzyme activity in the intestinal caecum (120x).

52 Anthelmintic efficacy of genistein
JPD : Vol. 28 (1), 2004
Fig.12: ATPase activity in the transverse cryostat section:
Diminished stain intensity in the dorsal tegument, somatic
musculature and the intestinal caecum after genistein treatment
(48x).
materials, but moderate activity was discernible in the
somatic musculature. Almost all the structures of the
treated flukes showed decrease in enzyme staining
intensity and a similar result was observed after
treatment with the reference drug (Figs. 3, 5, 6, 9, 12).
The results of the enzyme assays are described in Table
II. The AcPase activity in F. buski paralyzed with the F.
Fig. 13a- 13d: Protease enzyme activity in F. buski, fresh frozen
sections (photographs)
a
a: Control b: Control
c
c: Treatment with F. vestita
crude root peel extract
12
b
d
d: Treatment with genistein
vestita crude extract and genistein was inhibited by
42.75 % and 42 %, respectively, whereas oxyclozanide
caused a decline by 41.6 % compared to the controls.
Varying degree of inhibition of the AlkPase activity
(37.5-44.23 %) in comparison to control was observed
after treatment with the plant-derived components.
The ATPase activity also got reduced by 39.85% and
54.5% in treatments with crude peel extract of F.
vestita and genistein, respectively, while in the
reference drug-treated parasite a decrease by 36.6 %
was recorded. Biochemical analysis of 5'-Nu showed a
decrease in the enzyme activity by 47.8 % for F. vestita
and 51.9 % for genistein. The parasites treated with the
reference drug showed a decrease by 42.8 % (Table II).
Protease: The proteolytic enzyme activity was
demonstrable histochemically in both control as well
as the parasites treated with the various test materials,
though owing to the thickness of the sections, cellular
or subcellular localization of the protease activity was
not discernible. At the generalized tissue level, no
conspicuous change in the intensity of the enzyme
reaction was noticeable in the treated parasite from that
of the control (Fig. 13 a-d).
DISCUSSION
Ultrastructural studies have revealed that the tegument
in trematodes has an absorptive potential. The
processes of secretion and absorption appear to
operate concurrently in the gastrodermis of most
trematodes, although one or other function may
predominate at any given time. In a number of
helminth parasites AcPase, AlkPase, ATPase and 5'-Nu
have been detected both histochemically and
biochemically, and found to be closely associated with
the tegument, subtegument, somatic musculature, gut
and cuticle (Pappas, 1988; Leon et al., 1989; Kwak and
Kim, 1996; Pal and Tandon, 1998; Fetterer and
Rhoads, 2000). As revealed in the present study,
alterations occurred in the enzyme activity of the
parasite after treatment with the crude root tuber peel
extract of F. vestita and its active component genistein,
both showing synchronous effectiveness.
Oxyclozanide showed more or less similar effect as the

JPD : Vol. 28 (1), 2004
Anthelmintic efficacy of genistein
53
plant-derived components. After treatment, AcPase since studies on several epithelial cell types of
showed less stain intensity compared to the controls. mammals indicate that the transport systems and sites
Similar observations were made in various trematode of phosphatase activity are separate (Lumsden, 1975).
species exposed to treatment with hexachlorophene Non-specific AlkPase activity was localised at the
(Gupta and Sharma, 1973). Likewise, thiabendazole tegumental plasma membrane of Cyathocotyle, and in
and mebendazole changed the activity of AcPase in N. the surface channels of the tegument of Schistosoma
brasiliensis (Mishra and Srivastava, 1990). As in (Ernst, 1976). These findings (of AcPase and AlkPase
mammals, in helminths also, AcPase is usually in tegument and gastrodermis) raise the possibility that
associated with lysosomes and AlkPase is regarded as the tegument in some trematodes may function in a
indicative of membrane transport mechanisms digestive-absorptive capacity, analogous to that of the
(Barrett, 1981). The inhibition of AcPase activity by mucosal lining of the vertebrate intestine. Delabrethe
plant-derived components observed during the Defayolle et al. (1989) reported inhibition of AlkPase
present investigation is suggestive of the fact that activity by 23% and complete inhibition of glucose
absorption and intracellular digestion of drugs may uptake in Echinococcus multilocularis metacestodes
involve lysosomes (Colam, 1971). The lessening of following treatment with isatin, a known phosphatase
enzyme activity in the present study may probably be inhibitor, in vivo and attributed the depletion in the
due to its leakage into the medium as a result of the enzyme activity to the failure of glucose uptake
disruption of the absorptive surface (Hart et al., 1977). (Pappas, 1983). Some plant extracts also showed a
The major AcPase activity in trematodes is localised at
decrease in both AcPase and AlkPase activities in
the luminal surface of the intestinal caeca and is
Paramphistomum cervi in vitro (Chopra et al., 1991),
associated with the plasma membrane of the lamellar
but Fallon et al. (1994) showed more than two-fold
folds or the microvilli (Threadgold and Brennen,
increase in AlkPase activity in the PZQ-treated S.
1978). It is possible that the activity at these sites is
mansoni and attributed this increase to drug-induced
related to digestion of phosphate esters at membrane
tegumental damage exposing the normally concealed
level and in common with the phosphatases of the
enzymes on the tegumental surface of the worm.
mammalian intestinal brush border yields a kinetic The present investigation revealed a high ATPase
advantage in the subsequent absorption of the activity in the control worm. A tegumental
digestive products. involvement in ionic and water regulation in F.
In F. buski though histochemically AcPase showed
hepatica was indicated by the demonstration at the
+ +
higher stain intensity compared to AlkPase,
tegumental plasma membranes of Na /K -dependent
biochemical results showed an increased AlkPase
ATPase activity (Threadgold and Brennen, 1978). The
activity in the tissues compared to AcPase. In the
localization of ATPase in the somatic musculature of
present study AlkPase activity decreased by 37.5 to
F. buski indicates that the enzyme occurs in the
44.23% after treatment with the various test materials,
myofibrils. The ATPase activity was inhibited by 36.6-
and in histochemical localization also it showed
54.5% in the flukes treated with all the test materials.
decreased stain intensity. AlkPase has been localized
Similar observations were also made for E.
on the surface of both male and female Schistosoma
multilocularis isolated scolices of hydatids after
mansoni as a result of praziquantel-induced
exposure to mebendazole, thiabendazole, levamisole
tegumental damage (Mehlhorn et al., 1981; Modha et
and acrisaline (Benediktov, 1980). Drugs like
al., 1990). Any direct involvement of phosphatases in
phenathiazines, chlorimazine, diethylcarbamazine
absorption, as suggested by their association with
and centperazine caused significant inhibition of
absorptive structures in trematodes, seems unlikely
ATPase of the filarial parasite, Setaria cervi,

54 Anthelmintic efficacy of genistein
JPD : Vol. 28 (1), 2004
indicating thereby that this enzyme system could be a Aggarwal R, Sanyal SN and Khera S. 1992. Effect of anthelmintics
common target for the action of anthelmintic drugs
on phosphatases in Ascaridia galli. Acta Veterinaria Hungarica.
(Agarwal et al., 1990).
40: 243-249.
Barrett J. 1981. Biochemistry of Parasitic Helminths. Macmillan
In the present study the 5'-Nu activity decreased by Publishers Ltd, London, Basingstoke.
about 42-52% following treatment with genistein or Benediktov II. 1980. The effects of anthelmintics on ATPase and
other test materials in F. buski. 5'-Nu is supposed to dehydrogenase system' in scoleces of Echinococcus
have an active role in transport across plasma multilocularis. Naukova Dumka. 69.
membrane or may be involved with other enzymes in Buchmann K. 1998. Histochemical characteristics of
the hydrolysis of nucleosides to pyrimidine and purine Gyrodactylus derjavini parasitizing the fins of rainbow trout
(Onchorhynchus mykiss). Folia Parasitologica (Praha). 45 :
bases in parasitic nematodes (Barrett, 1981; Walter
312-318.
and Albeiz, 1985).
Bunitian HC. 1970. Deamination of nucleotides and the role of
In the present investigation protease activity was their deamino forms in ammonia formation from amino acids.
demonstrated histochemically in F. buski, both control In: Handbook of Neurochemistry, Lajtha, A (ed.). Plenum, New
and treated and there was no discernible change in the
York.
enzyme activity in the parasite exposed to the various Chopra AK, Sharma MK and Upadhyay VP. 1991. Effects of
ayurvedic anthelmintics on phosphatase activity of
treatments. The protease activity in Schistosoma is
Paramphistomum cervi. Indian Journal of Helminthology. 43:
reduced by specific trypsin inhibitors (Zussman and 65- 69.
Bauman, 1971). As demonstrated with histochemical
Colam JB. 1971. Studies on gut ultrastructure and digestive
and immunocytochemical techniques a major source physiology in Cyathostoma lari (Nematoda: Strongylida).
of proteolytic activity is the intestinal caeca and Parasitology. 62: 273-283.
protonephridia in trematodes (Howell, 1973; Dalton JP, Neill SO, Stack, C, Collins P, Walshe A, Sekiya M. et
Rotmans, 1977; Skelly and Shoemaker, 2001).
al. 2003. Fasciola hepatica cathepsin L-like proteases: biology,
function, and potential in the development of first generation
The alterations in the activity of the tegumental liver fluke vaccines. International Journal for Parasitology. 33:
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plant-derived components suggest that these enzymes Delabre-Defayolle I, Sarciron ME, Audin P, Gabrion C, Duriez T,
(excluding protease) may be a potential target of action Paris J and Petavy AE. 1989. Echinococcus multilocularis
for genistein, which, besides acting transtegumentally,
metacestodes: Biochemical and ultrastructural investigations
may as well affect through the gastrodermal interface.
on the effect ofisatin (2-3 indolincdionc) in vivo. journal of
Antimicrobial Chemistry. 23: 237-245.
ACKNOWLEDGEMENTS
Ernst SC. 1976. Biochemical and cytochemical studies of alkaline
phosphatase activity in Schistosoma mansoni. In: C.P. Read
This investigation was supported by a grant from Govt.
memorial Rice University Studies, vol. 62: pp 81-95.
of India under the DRS-III programme to the Fallen PG, Smith P, Nicholls T, Modha J and Doenhaff MJ. 1994.
Department of Zoology, North Eastern Hill
Praziquantel-induced exposure of Schistosoma mansoni
University, Shillong.
alkaline phosphatase:drug-antibody synergy which acts
preferentially against female worms. Parasite Immunology. 16:
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Journal of Parasitic Diseases
Vol. 28 (1) June 2004, pp. 57-60
Short Communication
Prevalence of phthirapteran ectoparasitic insects on
domestic hens of Rampur (U.P.)
A. K. SAXENA*, SANDEEP KUMAR, NIDHI GUPTA AND S.K. SINGH
Department of Zoology, Government Raza Postgraduate College, Rampur (U.P.) 244 901, India
As many as 60.9% of the fowls (510) examined in twelve localities of district Rampur (during July 2000 to
August 2002) were found infested with one or other kind of Phthiraptera. The order of prevalence of seven
species has been found to be Menopon gallinae (51.3%) > Goniocotes gallinae (25.4%) > Lipeurus lawrensis
tropicalis (15.8%) > Lipeurus caponis (11.5%) > Menacanthus cornutus (8.1%) > Goniodes dissimilis (7.9%) >
Lipeurus heterographus (6.9%). Significant positive correlation existed between mean monthly prevalence
rate and mean monthly temperature as well as photoperiod. The prevalence rate was significantly higher on
birds having poor health and poor plumage.
Keywords: Ectoparasite, Mallophaga, Phthiraptera, Poultry lice.
he prevalence and infestation intensity of
Tdifferent phthirapteran ectoparasites on certain
avian hosts viz. sparrows (Hoyle, 1938; Woodman and
Dicke, 1954), starlings (Boyd, 1951), blackbirds
(Baum, 1968), auks (Eveleigh and Threlfall, 1976),
procellariiform birds (Fowler et al., 1984), wood
ducks (Thul, 1985), reed bunting (Fowler and
Williams, 1985), Wilson's petrels and storm petrels
(Fowler and Price, 1987), leach's petrels (Fowler and
Hodson, 1988), manx shearwaters (Fowler and Shaw,
1989), five shorebird species (Hunter and Colwell,
1994), house martins (Clark et al., 1994), swifts (Lee
and Clayton, 1995), flycatchers (Potti and Merino,
1995), peacocks (Stewart et al., 1996), spanis raptors
(Perez et al., 1996), bee eaters (Kristofik et al., 1996;
Hoi et al., 1998; Darolova et al., 2001) have been
noted by selected workers. Only a few workers have
examined birds belonging to different orders from this
point of view (Ash, 1960; Klockenhoff et al., 1973).
Rekasi et al., 1997 and Rozsa, 1997 have discussed the
pattern in abundance of avian lice.
The prevalence rate of phthirapteran ectoparasites on
* Corresponding Author
Indian birds is not well documented. (Chandra et al.,
1990; Singh, 1999). Trivedi et al. (1992) recorded the
prevalence and intensity of Phthiraptera on poultry
birds of Dehradun. Prevalence of poultry shaft louse,
Menopon gallinae on poultry birds of Garhwal was
noted by Saxena et al., (1995). Present studies deal
with the prevalence and intensity of infestation of
seven phthirapteran species (Menacanthus cornutus,
Menopon gallinae, Lipeurus caponis, L. lawrensis
tropicalis, L. heterographus, Goniocotes gallinae and
Goniodes dissimilis) on the poultry birds of Rampur
district during July 2000 to August 2002.
Several methods have been described for delousing the
infested birds (Clayton and Drown, 2001) but during
the present studies, the prevalence and infestation rate
was recorded by timed visual counts as the birds
belonged to the private owners / poultry keepers. The
surveyor had to record the prevalence and intensity
without causing any harm to the bird. Hence, the bird's
legs were tied with rubber band / thread and both the
wings were held by the assistant (in upright position).
Bird's feathers were deflected manually and the
presence of lice was observed with the help of
magnifying torch. Lice were taken out and placed in
vials containing 70% alcohol. Each infested bird was

58 Phthirapteran ectoparasitic insects on domestic hens of Rampur (U.P.) JPD : Vol. 28 (1), 2004
again subjected to 5 minutes observation (for each of birds carrying three species infestation was 16.9%.
species) to record the number of lice by coding system Only a small percentage (2.4%) carried four species
(1-25 lice VL, 26-50 lice L, 51-75 lice- M, 76 -100 lice infestation. Infestation by M. gallinae and G. gallinae
H, and >100 lice - VH). was the most favoured combination on birds carrying
two species. Likewise, the popular combination of
Out of 12 species reported to occur, only seven could
birds having three species was M. gallinae, G.
be recovered from the poultry birds of Rampur. The
gallinae, and L. lawrensis tropicalis. Infestation by
chicken body louse, M. stramineus (which is otherwise
more than four species was not observed on the poultry
very common poultry lice) was not detected on the
birds of Rampur.
poultry birds of Rampur. Another louse species (G.
gigas) reported from poultry birds of Dehradun An attempt has also been made to find the degree of
(Trivedi et al., 1992) was also not recorded. Out of 510 correlation between mean monthly lice prevalence of
poultry birds examined from 12 different localities the period July 2000 to June 2001 and the mean
during July 2000 to August 2002, 60.9% were found monthly temperature, photoperiod and RH of the
infested with one or the other kind of poultry louse. corresponding period. Mean monthly prevalence was
Species-wise prevalence and infestation intensity has 76.5% in July. It decreased to the level of 65.3% in
been shown in Table.
August and remained nearly constant in succeeding
four months (60.0, 55.6, 58.8 and 56.5% from
Analysis of the data indicates that 54.4% of male birds
September to December, respectively). Prevalence
carried infestation of one or the other phthirapteran
showed a sharp decrease in January (48.4%) and
species. Prevalence on female bird was 63.6%. Sex
reached the lowest level in February (47.0%).
related difference in the prevalence was not significant
Thereafter, it rose gradually in succeeding four months
at 0 .05 level of significance. The colour of host birds
till it reached the highest level in June 2001 (53.3, 75.0,
was also recorded. The prevalence rate on the four
80.0 and 90.0% from March to June, respectively).
types of birds (white, black, brown and mixed colour)
Significant positive correlation existed between mean
remained 36.6%, 60.8%, 72.3% and 73.1%,
monthly prevalence and mean, monthly temperature
respectively. The colour related differences were not
as well as photoperiod (r = 0.79 and 0.76 respectively).
found significant at 0.05 levels.
However, correlation with mean monthly relative
Out of the 60.9% infested birds, 17.9% carried single humidity (r = -0.50) was not found significant, at 0.05
species infestation. Maximum percentage of bird level of significance.
(23.9%) showed two species infestation. Prevalence
Table : Intensity of infestation of seven phthirapteran species on poultry of Rampur, during July 2000
to August 2002.
Species Prevalence Intensity of infestation
VL L M H VH
M. gallinae 51.3 5.5 13.9 8.4 9.6 13.9
G. gallinae 25.4 3.3 10.8 3.3 4.5 3.5
L. l. tropicalis 15.8 1.4 6.5 1.8 3.7 2.4
L. caponis 11.5 1.6 5.7 1.2 1.4 1.6
M. cornutus 8.1 2.4 4.3 0.8 0.4 0.2
G. dissimilis 7.9 2.2 5.7 - - -
L. heterographus 6.9 0.2 6.5 0.2 - -

JPD : Vol. 28 (1), 2004
Phthirapteran ectoparasitic insects on domestic hens of Rampur (U.P.)
59
The degree of harmfulness of different species of difference in prevalence were not significant. Most of
poultry lice (in terms of loss of weight, vitality and the workers did not find any correlation with feather
productivity of host birds) is quite variable. M. colour of host bird and the louse prevalence / intensity.
stramineus was not found upon poultry birds of the Studies relating to prevalence rate of lice (M. gallinae)
district Rampur, but prevalence of the other dreaded on domestic fowls of Garhwal (Saxena et al., 1995)
poultry louse (M. gallinae) was highest on the birds of also indicate similar results.
this area. Nearly, half of the examined hosts carried
Present studies further indicate that most of the heavily
this louse species (51.3%). Prevalence of this louse on
and very heavily infested birds encountered during the
poultry birds of Dehradun and Garhwal were noted as
present investigation carried M. gallinae, G .gallinae
44.7% and 69.3% respectively (Trivedi et al., 1992;
and also L. lawrensis tropicalis. Birds heavily infested
Saxena et al., 1995). However, the prevalence of
with M. cornutus and L. caponis were rarely found.
another haematophagous poultry louse, M. cornutus
On the other hand, any bird carrying heavy or very
(whose degree of harmfulness and disease
heavy infestation of G. dissimilis and L. heterographus
transmission capabilities have yet not been
were not observed during the survey work.
investigated) remained comparatively lower (8.1%).
The order of prevalence of phthirapteran species ACKNOWLEDGEMENTS
recorded by Trivedi et al. (1992) was
M. gallinae—44.7% > M. cornutus—40.4% >
The authors are thankful to the Principal, Govt. Raza
M. stramineus—26.2% > G. gallinae —19.2% >
P.G. College, Rampur for providing laboratory
G. dissimilis—14.3% > L. caponis—13.8% >
facilities.
L. lawrensis tropicalis—9.2% and > REFERENCES
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Ash JS. 1960. A study of the Mallophaga of the birds with
M. cornutus was quite high on poultry birds of particular reference to their ecology. Ibis. 102: 93-110.
Dehradun. The prevalence of G. gallinae and L.
Baum H. 1968. Biologie und okologie der Amselfederlouse.
lawrensis tropicalis have been found comparatively
Angew. Parasitol. 9: 129-175.
higher on the poultry birds of Rampur while that of L.
caponis and G. dissimilis remained comparatively
Boyd EM. 1951. A survey of parasitism of the starling, Sturnus
vulgaris L. in North America. J. Parasitol. 37: 56-84.
lower. Lastly, Trivedi et al. (1992) did not find L.
heterographus on the poultry birds of Dehradun.
Chandra S, Agarwal GP and Saxena AK. 1990. Seasonal changes in
a population on Menacanthus eurysternus (Mallophaga :
Selected workers have noted the impact of host sex on Amblycera) on the common Myna, Acridotheres tristis. Int. J.
prevalence of avian lice. There are conflicting reports
Parasitol. 20 : 1063-1065.
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
prevalent on male birds in case of sparrows (Woodman House martin (Delichon urbica (L.)) feather louse Brueelia
gracilis Nitzsch (Mallophaga : Ischnocera) in Lincolnshire,
and Dicke, 1954), black birds (Baum, 1968) and 45
U.K. Entomologist 113: 198-206.
species of variety of birds (Ash, 1960). However, no
Clayton DH and Drown DM. 2001. Critical evaluation of five
difference in louse prevalence with respect to host sex
methods for quantifying chewing lice (Insecta : Phthiraptera).
has been noted in case of alcids (Eveleigh and J. Parasitol. 87: 1291-1300.
Threlfall, 1976), domestic hens (in case of M.
Darolova A, Hoi H, Kristofik J and Hoi C. 2001. Horizontal and
gallinae)(Saxena et al., 1995) and European beevertical
ectoparasite transmission of three species of
eaters (Hoi et al., 1998). During the present studies, Mallophaga and individual variation in European bee eaters
sex related differences in the prevalence rate were not (Merops apisater). J. Parasitol. 87: 256-262.
found to be significant. Thus, it may be stated that Eveleigh ES and Threlfall W. 1976. Population dynamics of lice
some host factors may occasionally cause variation in (Mallophaga) on auks (alcidae) from Newfoundland. Can. J.
louse prevalence in some cases, but generally there is Zool. 65: 2998-3005.
no significant difference in prevalence with respect to Fowler JA and Hodson D. 1988. The Mallophaga of Leach's Petrels
host sex.
Oceanodroma leucorhoa from North Rona, Scotland. Seabird
11: 47-49.
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.
ectoparasites populations of Procellariiform birds in Shetland,
Perez JM, Martinez IR and Cooper JE. 1996. Occurrence of
Scotland. Seabird 7: 23-30.
chewing lice on spanis raptors. Ardeola 43: 129-138.
Fowler JA and Price RA. 1987. A comparative study of the
Potti J and Merino S. 1995. Louse loads of pied flycatchers: effects
Ischnoceran Mallophaga of the wilson's petrel Oceanites
of host sex, age condition and relatedness. J. Avian Biol. 26:
oceanicus and British storm petrel Hydrobates pelagicus.
203-208.
Seabird 10: 43-49.
Rekasi J, Rozsa L and Kiss BL. 1997. Patterns in the distribution of
Fowler JA and Shaw GJ. 1989. The Mallophaga of Manx
avian lice (Phthiraptera : Amblycera : Ischnocera). J. Avian
shearwaters Puffinus p. puffinus from Ynys Enlli, Wales.
Biol. 28: 150-156.
Seabird 12: 14-19.
Rozsa L. 1997. Pattern in the abundance of avian lice (Phthiraptera
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: Amblycera , Ischnocera). J. Avian Biol. 28: 249-254.
Mallophaga and acari on reed bunting occupying a communal
winter roost. Eco. Ent. 10: 377-383.
Saxena AK, Kumar A, Surman and Singh SK. 1995. Prevalence of
Menopon gallinae Linne. (Phthiraptera : Amblycera) on
Hoi H, Darolova A, Konig C and Kristofik J. 1998. The relation
poultry birds of Garhwal. J. Parasitic Dis. 19: 69-72.
between colony size, breeding density and ectoparasitic loads
of adult European bee eaters (Merops apiaster). Ecoscience 5: Singh SK. 1999. Ecology of phthirapterans infesting pigeon in
156-163. Dehradun. Ph. D. Thesis, H.N.B. Garhwal University, Srinagar
(Garhwal) pp.150.
Hoyle W. 1938. Transmission of poultry parasites by birds with
special reference to the “English” or house sparrow and Stewart IRK, Clark F and Petrie M. 1996. Distribution of chewing
chickens. Trans. Kansas Acad. Sci. 41: 379-384.
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sparrow. Trans. Wisconsin Acad. Sci. 43: 133-135.

Journal of Parasitic Diseases
Vol. 28 (1) June 2004, pp. 61-64
Stilesia daulatabadensis N.Sp. from Capra hircus
V.P. SHELKE* AND G.B. SHINDE
Department of Zoology, Dr. B.A. Marathwada University, Aurangabad - 431004, MS. India
The present communication deals with a new species Stilesia daulatabadensis from a goat Capra hircus. It
differs from all the known species of the genus with the characters : scolex globular with large, oval suckers,
mature segment squarish, broader than long, testes 11 in number (7 on poral side and 4 on aporal side) cirrus
slightly curved, ovary a single mass, medium, oval, vagina long, posterior or anterior to the cirrus pouch,
ootype small, round, ventrolateral to the ovary, paruterine organs, two in each mature segment, in a
developing stage.
Key words : Capra hircus, Cestodes, Par Uterine organ, Stilesia
Short Communication
he genus Stilesia was erected by Railliet in 1893, or posterior half of the segments, cirrus thin, slightly
Tfrom Ovis aries in Europe, Asia and Africa, as curved vas deferens medium, thin, may or may not be
Stilesia globipuntata. Later on 14 species are added to coiled.
this genus by various workers in the world. The present
1- Scolex
communication deals with the description of a new
2- Mature Segment
species as Stilesia daulatabadensis n.sp
1
3
3 - 2Gravid Segment Segment
Twenty specimens of cestodes, of the genus Stilesia
were collected from the intestine of a goat Capra
hircus. All cestodes were preserved in 4% formalin and
stained with Harris Haematoxylin. Drawings are made
with the help of camera lucida. All measurements are
in millimeters (mm).
Stilesia daulatabadensis n.sp. (Fig. 1,2,3)
Scolex medium, globular rostellum absent, suckers
large oval, arranged in two pairs, occupy major portion
of scolex, neck long, elongated.
The mature proglottids medium, thin squarish, broader
than long with blunt round projection at the anterior
and posterior corners of segments, acrespedote testes
small to medium, oval, 11 in number 7 on poral side
and 4 on aporal side bounded laterally by longitudinal
excretory canals, cirrus pouch medium, oval, anterior
* Corresponding Author
1
2
1.0 mm
0.5 mm
1
2,3
3

62 Stilesia daulatabadensis N.Sp. JPD : Vol. 28 (1), 2004
The ovary is medium, oval; a single mass, at 1/3rd to globipunctata. Later on the following 14 species are
1/4th from the lateral margin of the same on each side, added to this genus.
vagina thin tube, long, placed posterior or anterior to 2. S. vittata Railliet, 1896
the cirrus pouch, slightly wavy, reaches and opens into 3. S. hepatica Wolffhugel, 1903
the ootype.
4. S. okapi Leiper.1935
The genital pores are medium, oval, situated in the 5. S. leiper Kadam, Shinde and Jadhav, 1980
anterior half of the segments, the paruterine organs are 6. S. caballeroi Kalyankar,Deshmukh &
two in number, in each mature segment, in a
Hatwalkar,1981
developing stage, on each side, dumb-bell shaped, 7. S. southwelli Shinde, Jadhav and Kadam, 1982
placed laterally in central medulla.
The gravid segments are medium, squarish, almost 4 8. S.aurangabadensis M.A. Majid, G.B. Shinde, and B.V.
times broader than long with convex lateral margin,
Jadhav, l982
with two paruterine organs in each segment. 9. S. garhwalensis Malhotra S.K; and Capoor V.N. 1983
Table 10.S.kotdwarensis Malhotra S.K. and Capoor V.N. 1983
Measurements of S. daulatabadensis
11. S marathwadaensis Shinde, Jadhav and Phad, 1985
Parts Measurements (mm)
12.S. alli S.N. Borde and G.B. Shinde, 1999
Length Breadth 13. Stilesia yavalensis G.B. Shinde and A.T. Kalse 1999
Scolex 0.738 - 0.818 0.670 - 1.011 14. S. dhondagae S.B. Deshmukh and L.V. Shinde, 2001
Suckers 0.2,05 - 0.375 0.363 - 0.375 After going through the literature the worm under
Neck 0.767 0.112-0.214 discussion differs as follows :
Mature Segment 0.131 - 0.189 1.195 - 1.199
The worm differs from S. caballeroi in the latter
Testes 0.029 - 0.049 0.019 - 0.039 having 1-11 testes on each side, vas deferens
Cirrus Pouch 0.112 0.029 - 0.058 anterior to the testes, less dense bundle of
Cirrus 0.112 0.005 convolutions, cirrus pouch oval, vagina posterior to
Vas Deferens 0.194 0.005 - 0.010 cirrus pouch, paruterine organ spherical on
Ovary 0.087 - 0.092 0.072 - 0.097 postolateral surface and from the host Capra
Vagina 0.262 0.005
hircus.
Genital Pore 0.019 0.024 - 0.034 The present worm differs from S. kotdwarensis in
Para Uterine Organs 0.180 - 0.223 0.092-0.112 the latter having 1-12 in number, ovary small
Gravid Segment 0.150 - 0.184 0.762 - 0.781
spherical, paruterine organ small, 2 in number,
between dorsal and ventral logitudinal excretory
vessels from the host Ovis aries.
Type species Stilesia daulatabadensis n.sp.
Host
Capra hircus
It differs from S. alli in the latter having scolex
round or oval, mature segments small or squarish,
Habitat Intestine
testes medium round 11 in number ( i.e. 5+6 = 11,
Locality At Daulatabad,
6+5 =11 ) cirrus pouch medium situated in the
Tq. Khultabad, Dist. Aurangabad, M.S., India
anterior half of the segment, ovary large oval single
Date of collection 25th Febuary, 1996 mass, paruterine organ bag like oval two in each
The genus Stilesia was erected by Railliet in 1893, gravid segment, found in Capra hircus.
from Ovis aries in Europe, Asia and Africa, a Stilesia

JPD : Vol. 28 (1), 2004
Stilesia daulatabadensis N.Sp.
63
It differs from S. dhondagae in the latter having testes
8-10 in number, arranged in the two groups, cirrus
pouch small, oval, situated middle to the posterior side
of the segment, ovary distinctly bilobed, elongated
having 8-9 acini and from the host Capra hircus.
Hence the above noted characters are valid enough to
erect a new species for these worm and the name
Stilesia thapari n.sp. is proposed in honour of Dr. G.S.
Thapari, Ex - Professor and Head, Department of
Zoology, Lucknow University, who has contributed a
lot in our knowledge of Helminthology
Majid MA, Shinde GB and Jadhav BV. 1982. On a new species
Stilesia Railliet 1893 (cestoda :Thysanosomatinae
skrjabin,1933) from sheep at Aurangabad Marath. Univ. J. Sci.
(Nat. Sci.) XXI 14 : 37 - 39.
Malhotra SK and Capoor VN. 1983. On two new species of
cestodes (cyclophyllidea), Stilesia garhwalensis n.sp. from
goat and Stilesia kotdwarensis n.sp. from sheep of the Garhwal
region. India. Acta. Parasit. Pol. 28 : 399 - 406.
Raillet 1896 - Surquelques Parasites des clromadise. Compt. Rend.
Soc. Biol. 51 : 18 - 21.
Shinde GB, Kadam SS and Jadhav BV. 1982. On a new cestode
Stilesia southwelli n.sp. from goat at Aurangabad, India.
Marath. Univ J. Sci.
ACKNOWLEDGEMENTS
Shinde GB, Jadhav BV and Phad AN. 1985. Stilesia
The Authors are thankful to the Professor and Head of
marathwadensis n.sp. (Cestoda:Thysanosomatinae Skrjabin,1
933) from Capra hircus at Aurangabad. Riv Parasit 2(46) : 213-
Zoology Department. Dr. B.A.Marathwada 215.
University, Aurangabad, for providing necessary Shinde GB and Kalse AT. 1999. On a new tapeworm Stilesia
laboratory facilities.
yavalensis (Cestoda; thysanosomidae, Fuhrmann, 1907)
REFERENCES
Sp.Nov from Capra \hircus in India. Uttar Pradesh J. Zool., 19
(1):89-91, 1999
Bhalerao GD. 1 934. On the occurance of Stilesia vittata (cestoda) Wolffhugel K. 1903. Stilesia hepatica nov.sp.ein Bandwum aus
in ovines in India. Ind. J. Vet. Sci. and Anim. Husbn. 5(1 ) : 28- den Gallengangen von Schafen and Ziegen Ostafrikas, Berlin
29. Tiearazi. Wochenschr. 43:661 =665.
Borde SN, Shinde GB. 1999. One new species Stilesia alli n.sp.
(Cestode : Thysanosominae, Fuhmiann,1907) from Capra
hircus at Hasnabad Dist. Jalna, India. Uttar Pradesh J. Zool.,
19(3):189-191.
Deshmukh S.B. and Shinde L.V. 2001 - New tapeworm from Capra
hircus at Kaij dist. Beed (M.S.) India. Rivista di parassitologia,
vol. XVIII (LXII) : 171 - 175
Gough. 1911. A monograph of the tapeworms of subfamily.
Avitellinae, being a review of the genus Stilesia and an account
of the histology of Avitelline centri-punctata. Riv. Quart. J.
Micro. Sci. 56 : 317 - 383.
Kadam SS, Shinde GB, Jadhav BV. 1980. On a new species of
Stilesia railliet,1893 cestoda Thysanosomatinae)
skrjabin,1953, from sheep at Aurangabad. Biol - vol II No. 3 :
33 - 36.
Kalyankar SD, Deshmukh AL and Hatwalkar VM. 1981. A new
species of the genus Stilesia Riailliet, 1893
(Anoplocephaloidea : Cestoda) from goat, Capra hircus at
Aurangabad Biol. 3 (1 ): 51 - 52
Kumar S and Lal SS. 1986. Effect of implantation of Stilesia
globipuncNata (cestoda : Anoplocephalidae) scolex on the
host, intestine Ind. J. Parasit (1986), 10-20 ;161 -163.
Leiper 1935 - Report on the helminth parasites of the Okapi living
in the Society's Gardens Proc. Zool. Soc. London 1935, 11-12.
Yamaguti S. 1959. Systema Helminthum Vol. II. The Cestode of
Vertebrates. Interscience Publisher, New Yorkand London,
p:339.

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Reference - Journal
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Trager W and Jensen JB. 1976. Human malaria
parasite in continuous culture. Science. 193 : 673-675.
Cheever AW, Macedonia JG, Mosimann JE and
Cheever EA. 1994. Kinetics of egg production and egg
excreted by Schistosoma mansoni and S. japonicum in
mice infected with a single pair of worms. American
Journal of Tropical Medicine and Hygiene. 50 : 281-
285.
The name of the journal should either be given full or
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Reference - Books
Schaniz PM and Kagan IG 1980. Echinococcosis. In :
Immunological Investigations of Tropical Parasitic
Diseases. Houba.V (Edt.) Churchill Living stone, New
York. pp 104-129.

Reference-Conferences
Mahajan RC, Malla N, Stella M and Ganguly NK
1995. Cysticercosis in India: Immunodiagnosis and
Treatment. Paper presented at the Twelfth National
Congress of Parasitology. Panaji, Goa, India, 23-25
January.
Reference-reports
World Health Organization, 1984. The Leishmaniasis.
Technical Report Series No. 701. Geneva,
Switzerland.
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Forthcoming Scientific Events
Date/Location : September 2-4, 2004; Warsaw
Title : XX Congress of the Polish Parasitological Society
Information : Dr. Anna Rocka, Department of Medical Biology, Medical University of Warsaw,
Nowogrodzka Street 73, 02-018 Warszawa, Poland.
E-mail/Ph./Fax : lchomicz@ib.amwaw.edu. pl., Phone/Fax: (48-22)-625-2468
Date/Location : September 23-26,2004; Mumbai, India
Title : 50 Years of Medical Writing - International Conference on Journal Writing
and Publishing
information : Dr AtuI Goel. Department of Neurosurgery, Seth G. S. Medical College. Parel.
Mumbai -400 012. INDIA
E-mail/Ph./Fax : goldcon@jpgmonline.com.Phone: 91-22-24129884. Fax: 91-22-25032398
Date/Location : September 26 - October 8, 2004; Montevideo, Uruguay.
Title : Course on Pathogen Trypanosomes - Mammalian Host-cell
Interactions: Biochemistry, Cell Biology And Prospects For Drug Development
Information : Center for Free Radical and Biomedical Research (CFRBR). Departamento de
Bioqulmica. Facultad de Medicina. Universidad de la Republica, Montevideo. Uruguay.
Website : www.hhmi.org/grants/awards/inst/intl_courses.html
Date/Location : September 27-29,2004; Boston
Title : XXXVI Annual Society for Vector Ecology (SOVE) Conference
Information : Major Dhillon
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Website : www.sove.org.
Date/Location : September 28-29,2004; Boston
Title : American Society of Tropical Medicine and Hygiene (ASTMH), Intensive
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E-mail/Ph./Fax : astmh@astmh.org:; Phone: 847/480-9592; Fax: 847-480-9282
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Date/Location : September 30 - October 3, 2004; Boston
Title : XLII Infectious Diseases Society of America (IDSA) Annual Meeting
Information : IDSA, 66 Canal Center Plaza (Suite 600), Alexandria, VA22314
E-mail/Ph./Fax : Fax: 703/299-0204
Website : www.idsociety.org

Date/Location : October 7 -8, 2004
Title : Vaccines 3: Frontiers in vaccine development Institut Pasteur
Information : www.pasteur.fr/applications/euroconf/vaccines3/index.html
E-mail/Ph./Fax : euroconf@pasteur.fr ; Fax: 33()1 40 61 3025
Date/Location : October 30 - November 2. 2004; Washington. DC
Title : IVL Inter science Conference on Antimicrobial Agents and
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Information : ASM Managed Meetings, 1752 N Street, NW, Washington. DC 20036-2904.
E-mail/Ph./Fax : conferences@asmusa.org: Phone: 202/942-9261: Fax: 202/942-9340;
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Date/Location : November 7 - 11, 2004; Miami
Title : Llll American Society of Tropical Medicine and Hygiene (ASTMH)
Annual Meeting
Information : ASTMH Secretariat. 60 Revere Drive (Suite 500), Northbrook, IL 60062
E-mail/Ph./Fax : astmh@astmh.org; Phone: 847/480-9592; Fax: 847/480-9282
Website : www.astmh.org
Date/Location : November 12 - 14, 2004; Galveston, Texas
Title : McLaughlin Symposium - The Changing Landscape of Vaccine Development:
Translating Vaccines for Emerging Diseases and Biodefense to the Marketplace
Website : www.utmb.edu/scvd. ( Note: For additional information, see PDF file.)
Date/Location : November 14 - 20, 2004; Southampton, Bermuda
Title : American Society of Microbiology (ASM) Conference on "DNA Repair
and Mutagenesis"
Information : ASM Managed Meetings. 1752 N Street. NW, Washington, DC 20036-2904
E-mail/Ph./Fax : conferences@asmusa.org; Phone: 202/942-9261; Fax: 202/942-9340
Website : www.asm.org/ meetings/index.
Date/Location : Nov 17-20, 2004
Title : 5th Louis Pasteur Conference on Infectious Diseases Institute Pasteur
E-mail/Ph./Fax : clp@pasteur.fr
Website : www.pasteur.fr/infosci/conf/sb/CLP5/
Date/Location : July 10-13, 2005; Singapore
Title : V International Conference on "Urban Pests (ICUP)
Information : ICUP 2005 Secretariat, 73 Bukit Timah Road, Rex House (Suite 03-01),
Singapore 229832
E-mail/Ph./Fax : info@icup2005.com.sg; Phone: (65)-6330-6830; Fax: (65)-6336-2123;
Website : www.icup2005.com.sg.

Date/Location : September ??-??, 2005; Marseilles
Title : XVI International Congress for "Tropical Medicine and Malaria and
Centenary of Tropical Medicine Institute for the French Army"
Website : www.iftm.org.
Date/Location : December 11 - 15, 2005; Washington, DC
Title : LIV American Society of Tropical Medicine and Hygiene (ASTMH)
Annual Meeting
Information : ASTMH Secretariat, 60 Revere Drive(Suite 500), Northbrook, IL 60062
E-mail/Ph./Fax : astmh@astmh.org; Phone: 847/480-9592; Fax: 847/480-9282
Website : www.astmh.org.
CONGRATULATIONS!
Prof. Md. Hafeez, Deptt. of Parasitology, College of Veterinary
Science, ANG Ranga Agriculture University, Tirupati has been awarded
the distinction of "Environmentalist of year award-2003" by the
'International Board of Awards of "National Environmental Science
Academy", New Delhi. The award was presented to him at XVII Annual
Conference of the Academy and International Workshop on 'Recent
Trends in Environmental Sciences and Eco Journalism', held in New
Delhi from 24 -26 April, 2004.

Third National Workshop on
"Simple Diagnostic Methods in Infectious Diseases"
7th -11th December 2004
Department of Microbiology
Jawaharlal Institute of Postgraduate Medical Education & Research,
Pondicherry
The number of Participants will be restricted to 20
The Last date of receipt of applications is 30th September 2004
For details please contact
Course Coordinator:
Dr. Subhash Chandra Parija MD, PhD
Professor & Head
Department of Microbiology, JIPMER
Pondicherry - 605006
Telephone: Off: 0413-2272380-90, Extn.:3200,
Residence: 0413-2253016; Mobile: 0413 3114418
Telegram: JIPMER, Fax: 0413-2272067
E-mail: idworkshop@rediffmail.com
The Selected Participants have to submit a registration fee of Rs. 1,500/- (Rupees One
thousand and five hundred only) for the workshop and may be sent in as a bank draft payable to
"Course-Coordinator, Infectious Disease Workshop" at Pondicherry.
Note: The cost of boarding and lodging is not included in the registration fee. Accommodation
will be provided in JIPMER guesthouse on a double room-sharing basis. The selected
participants have to pay an additional Rs. 1,000/-(Rupees One thousand Only) towards
accommodation and boarding expenditure and is mandatory.
For further details about the workshop log on to the web site www.jipmer.edu

JOURNAL OF PARASITIC DISEASES
Volume 28 Number 1 June 2004
CONTENTS
PRESIDENTIAL ADDRESS
The Perfect Match - Parasite & host: Made for each other 1
M.S. Jairajpuri
REVIEW
Role of DNA microarray Technology for understanding differential 5
gene expression in Parasitic Diseases
A.Debnath, A. Sen, James H. Mckerrow and P. Das
EPIDEMIOLOGY
Epidemiological studies on bovine microfilariasis in coastal districts 17
of Andhra Pradesh
V. Pavan Kumar, B. Sreedevi, T. Venkata Reddy
and K. Nalini Kumari
Seasonal occurrence of helminth parasites in Schizothorax in 23
Dal Lake Kashmir
A.R. Khan, M.Z. Chishti, Fayaz Ahmad, Majidah Rashid and
Shafqat Bakshi
DIAGNOSIS
A monoclonal antibody to 120 kDa B. malayi antigen with 29
diagnostic potential in Bancroftian filariasis
Balaji Ganesh, B. Parab P.B., Katdare M., Reddy M.V.R.
and Harinath B.C.
CHEMOTHERAPY
Anthelmintic efficacy of extract of Stephania glabra and aerial root 37
extract of Trichosanthes multiloba in vitro: two indigenous plants in
Shillong, India.
V. Tandon, L.M. Lyndem, P.K. Kar, P.Pal, B. Das and H.S.P. Rao
Anthelmintic efficacy of genistein, the active principle of 45
Flemingia vestita (Fabaceae): Alterations in the activity of
the enzymes associated with the tegumental and gastrodemal
interfaces of the trematode, Fasciolopsis buski
Pradip Kumar Kar and Veena Tandon
SHORT COMMUNICATION
Prevalence of phthirapteran ectoparasitic insects on domestic hens 57
of Rampur(U.P.)
A.K. Saxena, Sandeep Kumar, Nidhi Gupta and S.K. Singh
Stilesia daulatabadensis N. Sp. from Capra hircus 61
VP Shelke and GB Shinde
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