This is the lecture to be delivered in the WINTER SCHOOL on MOLECULAR BIOLOGICAL APPROACHES FOR DIAGNOSIS AND CONTROL OF
PARASITIC DISEASES at IVRI , Izatnagar in December ,2013. I am posting it on my blog for wider circulation among scientific community, who are interested in schistosomiasis: will be pleased to see their comments.
M C AGRAWAL
FORMER NATIONATIONAL FELLOW AND EMERITUS SCIENTIST
COLLEGE OF
VETERINARY SCIENCE AND A H , JABALPUR 482001
drmcagrawal@gmail.com www.indianschistosomiasis
.blogspot.com
(Present address : ¾ Datt Arcade Phase Three, South Civil
Lines, Jabalpur 482001)
Those who are working on
diagnosis of schistosomiasis are aware how vast is the subject and its
impossible for anyone to cover the topic in one hour lecture. Therefore, it
will not be my goal to cover the whole subject , rather I will discussion some
pertinent points on the topic at this occasion. We have discussed in detail about
the diagnosis of schistosomiasis – both parasitological and immuno-diagnosis in
our recently launched book(Agrawal,2012)
,Schistosomes and schistosomiasis in
South Asia (ISBN 9788132205388 www.springer.com) and you may refer this
book for all the detailed information and related discussion . One advice is to
those scientists who are interested to work on any problem of schistosomiasis
in south Asia is to study well all the concerned literature on the subject (as it is too vast )
prior undertaking any work as this prior study will inform them about the work already done and the existing problems;
such exercise will avoid duplication of work and will help in understanding
well the present problem related to south Asian schistosomes and
schistosomiasis which are certainly different from African or East Asian
schistosomiasis .
Generally we are working and discussing methodology of one or other
diagnostic technique , its sensitivity and specificity , but there are no
discussions whether we need to devote
our time , energy and funds for developing that specific diagnostic technique and what will be
its utility in present scenario. I think such discussion is necessary and my this
lecture will focus on this topic while discussing schistosome diagnosis.
Further, by definition- as per
Oxford dictionary , diagnosis means “
statement of nature of a disease or
other condition made after observing its signs and symptoms” . Thus strictly diagnosis
was restricted to what we call clinical diagnosis and as you know with passage
of time it has gone in the back ground and laboratory diagnosis has taken a
central stage-so is the case with schistosomiasis .One reason for this is our
realization of difficulty in identifying the disease on symptoms alone due to
commonness of them in many other
ailments. Therefore, we cannot whisk away laboratory diagnosis and in fact this
is the main topic while discussing schistosomiasis diagnosis. To this , I have taken liberty to include
identifying schistosome infection in snail host ,as well- this is to understand
the subject and related problems quite well.
OUR AIM OF DIAGNOSIS : The basic question is what is our aim of
identifying schistosome infection ? Whether it is only for treating a particular
animal coming to veterinary hospital ? Or to declare a geographical area
endemic with a given schistosome species ? Or to understand epizootiology of
the infection ? Or to study zoonotic aspect of a schistosome species ? Or to
know percentage of infected animals in each species ,age and sex wise ? Or to
study immune status of animals after vaccination program ( so far ,no
commercial vaccine is used against schistosomiasis in India ; it may be only on
experimental basis ) . Or we wish to collect schistosomes during necropsy or in
experimental animals for molecular and other studies . Again schistosomiasis is existing in animals
in two important forms (nasal schistosomiasis or snoring disease and
hepato-intestinal schistosomiasis ) and diagnosis of both are different . So,
there are varied aims of diagnosis hence our methodology of diagnosis should
also differ as per our aim . Therefore, I will discuss the subject as per our
aims and what best methodology we should follow to attain our aims of studies.
SENSITIVITY AND SPECIFICITY OF TESTS :
While starting the discussion,
let me clarify that diagnosis of schistosomiasis is difficult due to two
reasons . First, the symptoms in hepatic form like diarrhea, dysentery,
anorexia, weakness, bottle jaw etc are
also present in other chronic or sub-acute infections . However, in snoring
disease , the animal exhibits pathognomic signs like snoring sound, nasal
discharge and nasal granuloma but totally depending on these symptoms may miss
the infection in buffaloes or some cattle which are passing eggs in nasal
discharge but without symptoms; and by this strategy , you cannot identify
nasal schistosomiasis ,as is existing in Jabalpur , where local cattle are
resistant to Schistosoma nasale but
buffaloes are harboring the parasite- this was confirmed by Banerjee and Agrawal (1991).
Therefore, for confirming the
infection, we have to depend on laboratory diagnosis which is of two types
–Parasitological and immunological- and both are having their own problems
which have been discussed in chapter 7 and 8,respectively in above book. Basically
, laboratory diagnosis is difficult due to poor sensitivity with no ante-mortem
parasitological method being capable to correctly estimate schistosome prevalence
in a given geographical area . For these reasons, I have argued about under
estimation of schistosomiasis in whole of south Asia ( Agrawal, 1999)
OUR
PROBLEMS IN PARASITOLOGICAL METHODS
In parasitological diagnostic
method , we can deal only with faeces (in hepatic form) of the animal during ante-mortem period . The most common method of faecal
examination, followed in any veterinary diagnostic laboratory, is direct wet
smear method . In some advance laboratories
sugar flotation or salt concentration method is also followed. Ideally ,
both supernatant and sediment should be examined for checking nematode and
fluke infections but many times only supernatant is examined without caring for
the sediment. Ironically , examination
of faeces by both these methods have resulted in identifying only 2-8%
infection in any animal species whereas ,in reality , the infection rate was
varying between 60-80% . Obviously, such diagnostic method cannot be
recommended for diagnosing schistosomiasis when surveillance of the infection
is our aim.
ANTEMORTEM DIAGNOSIS
Therefore, my laboratory assessed
different coprological methods which may be used preferentially under field
conditions. We have tried and compared direct smear, salt floatation, acid-ether or formal-ether, sieving method,
and Kato-katz methods in experimentally infected laboratory animals as well as in field animals (Agrawal 2000 , Gupta 2002 ,
Vohra 2005 ). The results varied as
per animal species, age of animal and intensity of the infection but
sensitivity of these tests ascended from
direct smear to salt floatation, kato-katz , formal ether methods leading highest level to 16% .
As any of the above egg detection
method could not provide a practical solution of diagnostic problem, we tried
hatching method by taking advantage that the eggs of schistosome species
contain fully developed miracidium ,
ready to hatch ,soon after coming in contact with fresh water . There are many factors which
influence this hatching of eggs and have been discussed elsewhere (
Agrawal,2012).
In summary , we can say that
hatching method proved always superior over any egg detection method hence this
is the test which we recommend to be followed in field as well as in research
laboratories. When we studied the method more meticulously , it was
found more effective than any egg detection method where number of schistosome
eggs in the faecal material was low- this is the young or old animals where egg
concentration is low. For instance ,the hatching method ,though showed higher
prevalence in pigs in comparison to egg detection methods, could not show
significant variation in prevalence rate of porcine schistosomiasis at Jabalpur
.This was because of higher faecal
concentration of schistosome eggs in pigs (in comparison to other animal
species) resulting its identification by other egg detection methods, as well.
The other animal species where we observed different
results, while comparing miracidia detection vis a vis egg detection , was experimentally infected albino mouse.
Here, acid-ether method was more simple and sensitive than hatching method
which failed many times to detect miracidia in the faecal material.
The hatching method is not only
simple but also cost effective as it does not require any chemical or use of
micro slides. Therefore, this is an ideal method that can be followed under
field conditions with little efforts .
POST MORTEM METHODS :
Even diagnosing of
schistosomiasis is not easy while conducting post mortem of an animal in
comparison to other helminthic infections. Thus a simple cut in the liver may
yield liver flukes, opening of rumen reveals amphistomes while intestine lumen
will cause recovery of many nematode species . And in all these organs, you
will not get schistosomes declaring the animal negative for schistosome
infection. I may substantiate my statement by one reference of Rathor (1998)
who compiled postmortem reports of more than eighteen thousand buffaloes of 28
livestock farms of India; the buffaloes were harboring Fasciola and amphistome infections but no case of schistosomiasis
was recorded in these post mortem reports . As intermediate hosts of amphistomes and schistosomes are identical ,it
is difficult to believe that all the animals were negative for
schistosomes-rather this is a case of failure of diagnosing schistosomiasis in
any of these animals due to following routine post mortem techniques.
This issue was attended by us and
we have described specific techniques which should be followed during
conducting post mortem of an animal for diagnosing schistosomiasis in them .
These are two techniques ; one is perfusion technique for recovering alive
blood flukes and the other is egg detection method using intestinal scrapings
or liver pieces (Agrawal 2012, Vohra
2005 )
IMMUNO-DIAGNOSIS :
A lot of research work globally is being carried out on immuno-diagnosis
mainly to study human schistosomiasis in
endemic countries and there are various reviews over the subject. It is
difficult to cover them in one hour lecture . Nevertheless, I will mention some
important facts which will help our discussion . For more details , you may
read chapter 8 of the above referred book (Agrawal,2012).
ANTIBODY DETECTION METHODS :
In the beginning of the work, all
the efforts were made to detect specific schistosome antibodies, developed by
the host due to schistosome pre-
infection by using different types of the antigens .
There was fascination to use live
larval stages of the blood flukes ,all three forms- viable eggs, miracidia, and
cercariae- as experimental works have shown development of precipitates or
blebs around viable schistosome eggs or immobilization of miracidia or development of a hyaline membrane around
cercariae when these larvae were immersed in the serum containing schistosome
antibodies. The former two larvae were discarded as the tests were not very
sensitive while CHR (cercarian hullen reaction ) proved a promising
immunodiagnostic test though with the requirement of procuring alive cercariae
for conducting the immunodiagnostic test.
To avoid the requirement of alive
larvae, soluble antigens were developed using different methods and different
stages of the schistosomes. Those who dealt the subject realized how difficult
it is to recover a good quantity of schistosomes comparatively due to its
smaller size and its location in the
blood vessels . Therefore, soon ,the immuno-diagnostic tests requiring larger
quantity of antigens i.e. ring precipitation test were discarded . And in
recent years ,all attention is diverted on ELISA ; as plate ELISA requires
larger quantity of antigen and is more tedious , Dot ELISA is the method of
choice among schistosomologists as each test requires only nano gram of
antigens and is more simple to perform .
Looking to importance of
diagnosis of fluke infections ,a NATP project was run by ICAR with main center at IVRI during 2000-2004 and making Jabalpur, Hisar, as investigating centers
for diagnosis of schistosomiasis ; some important work was also carried out at
Bangalore veterinary college. Though these centers were able to develop Dot
ELISA tests for diagnosing schistosomiasis with higher sensitivity , specificity
could not reach to higher levels . Even some scientists ( Sumanth et al 2003) found Dot ELISA positive in all the field animals
who ever have been exposed to schistosome infections (?) and presently were
either negative or positive for
schistosome eggs. What will be the utility of such diagnostic test in studying
schistosomiasis remains debatable (Agrawal
2012).
ANTIGEN DETECTION METHOD :
The antibody detection methods
have now been discarded globally by scientific community due to four reasons. First , it does not differentiate
between present and past infection . Second, it fails to calibrate intensity of
the infection ; neither it reflects that all these positive animals are immune
to re-infection . Third, it is difficult to collect blood from large animals
under field conditions where controlling of the animals is a great problem (now
there are ethical questions for collecting human blood merely for surveillance
work). Fourth,the invasive method (collecting blood) merely for diagnosis is
not favored due to the chances of transmitting viral and other infections; this
has become more controversial in human cases where there are chances of
transmitting HIV due to minor negligence of the technician.
Therefore, in recent years ,
attention has been diverted to detect schistosome antigen in faeces or urine of
man and animals and principles of ELISA are followed for developing these
methods. To make diagnostic test more specific and sensitive monoclonal
antibodies have been used to attach them on nitrocellulose strip. Again ,
leading to field adaptation of these
tests , single step diagnostic kits have been developed where a solution is to
pour on the strip which will develop two bands in case of positivity and only
control band if the case is negative for schistosome infection.
Let me clarify that no commercial
single step diagnostic kit is developed or available in India , though needed
at least for diagnosing migratory human cases . This has been developed in some
other countries and one single step
dipstick kit from European Veterinary Laboratory , Netherland was tried by us
resulting in five positive human cases who were either suffering or were having
history of cercarial dermatitis.
SCHISSTOSOMA SPECIFIC TESTS :
As this is the winter course on
molecular studies on diagnosis and control of parasitic infections, an important clarification , regarding all
immuno-diagnostic methods is that they are capable to diagnose Schistosoma
infection- only up-to genus level and none of these tests are species specific .
Therefore, it is important to note that none of these tests will inform which schistosome species is present
in positive host species (in one sense , the test is good being able to be
positive if the animal is infected by any schistosome species thus avoiding
checking with many species specific tests- if and when developed ). When it fails to identify schistosome
species, any of these tests are of no use in identifying strains or sub-species
of the schistosomes; neither they may help in detecting any new schistosome
species.
Not only immuno-diagnostic
methods, but our egg detection methods may fail to differentiate among
schistosome species where egg morphology is closely resembling. If you study
the literature, you will notice that
all oval shaped schistosome eggs could
not be ascribed to S.haematobium or S.indicum ; likewise , spindle shaped
eggs does not mean only presence of S.spindale
in the area ( Rollinson and Southgate
1987).
Therefore, now we have come to our main discussion –what is the aim of
our diagnosis ?
It would have been clear from
above discussion that the above methodology will not solve all our problems
related to schistosomiasis and we have to stitch specific methods of diagnosis
as per our requirements. I am discussing some of them in brief :
DIAGNOSIS FOR TREATMENT : When an animal with certain symptoms
visit to a veterinary hospital , the main aim is the treatment of that
particular animal. As the symptoms of schistosomiasis are common with other
infections, though treatment is not the same, the differential diagnosis for
schistosomiasis is important. The parasitological diagnostic tests have been
termed as ‘Golden tests’ hence it is
always recommended to diagnose these animals by coprological methods. This should
include acid-ether or formal –ether method along with hatching method .The
tests may be repeated twice or thrice if one time method shows negativity .
COLLECTION OF SCHISTOSOMES : If there is the need of collecting
alive schistosomes from any animal species, perfusion method is the ideal one
(Agrawal 2012). Recovery of schistosomes will also confirm presence of the
infection in that particular host species and the geographical area to which
the animal belonged . In old days, the
scientists teased the blood vessels for checking the animal positive for
schistosomiasis and collecting alive blood – flukes; this method is tedious as
well as less sensitive hence not recommended. Previously, it was difficult to
apply perfusion technique as it was carried out by the use of a peristaltic
pump which was costly and not available in Indian laboratories . however, we
have replaced it ,successfully, with a
vertical water pump, most commonly used in desert coolers in Northern India
(costing Rs 500/) hence now it is feasible to any laboratory to perform
perfusion technique for recovering schistosomes during necropsy of the animals
and the technique has been described in details in the book ( Agrawal,2012).
The alternative method, though
less sensitive , is to collect mesentery or nasal cavity (when working on nasal
schistosomiasis) of the suspected
animal, cut into small pieces and soak in Luke warm saline for 4-6 hours.
Afterwards, the saline is filtered using a muslin cloth which is transferred to a petri-dish to collect the blood flukes (
Agrawal,2012).
IDENTIFYING NEW ENDEMIC AREA : As you have come from different places of the
country, it may be interesting for you to confirm whether a geographical area
of your state or district or Tahseel is positive for any schistosome species.
For confirming this possibility, you have to examine both intermediate and
final hosts- in all seasons and in good number
else your results will not be reliable.
Till now only Indoplanorbis exustus and Lymnaea luteola have been identified as
intermediate hosts for all the five well studied schistosome species i.e. Schistosoma
indicum,S.spindale,S.nasale,S.incognitum and Orientobilharzia dattai while the snail host of Bivitellobilharzia nairi , O.bomfordi
(?), S.haematobium (?- Gimvi
infection) have still not been confirmed. Therefore, all the fresh water snails
of the area should be examined meticulously for shedding of brevifurcated ,
apharyngeal ,non-ocillate cercariae ;
presence of such type cercariae will indicate prevalence of any mammalian
schistosome species in the area (Agrawal,2012) .If these cercariae are shed by
a new snail species, there are all chances that you are dealing with a new
schistosome species .
As morphological studies of these
cercariae cannot differentiate different mammalian schistosome species and
certainly cannot confirm strains or sub-species of the schistosomes ,it is
recommended to develop molecular techniques to study these cercariae. So far, it is suggested that
the snails are infected only with mono-species of fluke infections and
molecular studies will be able to confirm or otherwise the above notion .
When you are examining final
hosts for schistosome infection, first check if the animal is not a migratory
animal otherwise origin of the infection will remain questionable. Therefore,
area of birth of the animal is important in such studies and only by this
methodology we suspected that Jabalpur is also harboring Schistosoma nasale in the buffaloes ( Banerjee and Agrawal 1991).
Though immuno-diagnostic methods
are having high sensitivity (80-95%), their specificity ranks between 60-70%
therefore solely depending on immuno-diagnosis while confirming a new endemic
area is not a very good idea. However, not utilizing any immuno-diagnostic method
may not reveal schistosome infection in a geographical area hence its use is
important while exploring possibility of schistosomiasis in any animal species.
It must also be associated with
sensitive parasitological methods which should always include hatching method.
While examining animals for S.nasale ,instead
of nasal discharge ,check the nasal scrapings as the latter is four times more sensitive than the former method .
STUDYING ZOONOTIC POTENTIALS OF SCHISTOSOME SPECIES : This is a topic of great interest in whole of
South Asia but has not been studied meticulously . Identifying an endemic focus
of urinary schistosomiasis in Gimvi village of Ratnagiri district, Maharashtra
state ( Gadgil and Shah,1952)
generated considerable interest in human schistosomiasis in the country but non
–detection of clinical urinary cases from other parts of the country created a
complacent view of absence of human schistosomiasis in other parts of the
country . This view was developed ignoring the fact that S.incognitum was first detected in two human cases by Chandler (1926) and there are reports
of finding schistosome eggs in human excreta in other parts of the country (Agrawal,2012) . The topic has been discussed
in more details while reviewing present status of schistosomiasis in India ( Agrawal,2005). Prevalence of cercarial dermatitis in rural India is very common (Agrawal,2012)
although no further studies have been carried out on the ailment. Thus we are not sure to what stage of
development the schistosomula reaches in humans showing different immunological
conditions .
In the above cases, generally
parasitological methods have failed to detect schistosome eggs in human excreta
,therefore, it is advocated to use a more sensitive and specific antigen
detection test . The specificity of single step diagnostic kits , developed by
using S.mansoni or S.haematobium, in other countries is not
very high warranting developing such kits using Indian schistosome
species. Likewise, we do not have any
specific immunological test for confirming cercarial dermatitis cases ; this is
only history of dermatitis and
recovering schistosome cercariae from the fresh water snails which is
collaborated with the ailment. Therefore, it will be beneficial to develop an
immunodiagnostic test which may confirm cercarial dermatitis- ideally with
possibility of differentiating cercarial dermatitis caused by avian schistosome
cercariae and that by mammalian schistosome cercariae.
As you will notice that the
single step diagnostic kit or immunodiagnostic test for cercarial dermatitis
will not be able to identify schistosome species or strains involved in each
case. Therefore, there is the need to develop molecular techniques for
identifying species or hybrids or strains of schistosomes ,existing in whole of
south Asia. Obviously, both the methodologies are supplementary to each one in
solving the problem of schistosomiasis in the region.
CHECKING HOST IMMUNITY AGAINST
SCHISTOSOMES : In viral and
bacterial infections , immunological tests have also been used to check immune
status of a given population and are good tools for vaccination programs . When
we look the scenario with relation to schistosomiasis , two facts have emerged
.
First, there is no vaccine which
is being used for controlling schistosomiasis . This is the case both for human
schistosomiasis as well as for animal schistosomiasis . More and more efforts
are being made to develop a vaccine which may protect the host against
schistosome infection. I am not aware if any institute in India is trying to
develop a feasible vaccine for schistosome infection in any host species.
Secondly, our immuno-diagnostic
tests are able to reveal presence of antibodies in positive cases but whether
presence of these particular antibodies
will be able to protect the host against re-infection with schistosomes
is questionable .So far , we have not developed any immuno-diagnostic test whose positivity also guarantee protection of
the host against re-infection.
SURVEILLANCE OF ANIMALS FOR SCHISTOSOMIASIS : Whenever , we
undertake surveillance work for identifying any infection, our diagnostic test
should be most sensitive and specific in diagnosing that infection. Ironically,
no diagnostic test stands on above criteria with regards to schistosomiasis . With Indian schistosomes , parasitological
tests are least sensitive due to comparatively
lower egg excretion hence their
sole use in surveillance work is never advocated . In our opinion, hatching
method has proved most sensitive
parasitological method during
ante-mortem hence must be included in all surveillance works. Those cases,
positive by hatching method, must also be examined by a more sensitive egg
detection method which will help in
identifying schistosome species of that area ,as well.
Since parasitological tests are
least sensitive, the surveillance work must be carried out by supplementing
parasitological methods with a more sensitive and specific immuno-diagnostic
method. All the work , carried out in India, have proved Dot ELISA as most
promising immuno-diagnostic test hence this should be incorporated in
surveillance work but fully understanding its limitations (Agrawal,2012).
The antigen detection test in
animal faeces will be more simple and be able to detect existing schistosome positive
cases hence there is the need to develop these tests for understanding
schistosomiasis in South Asia.
As we have already discussed the
limitations of these immuno-diagnostic tests as well as those of
parasitological tests, schistosomes and schistosomiasis cannot be understood
well in whole of south Asia without developing more sophisticated molecular
techniques which will help in identifying new schistosome species and also
hybrids of the schistosomes which have developed in the continent due to hybridizations
with existence of two or more schistosome species in a single host species. A
collaboration with some international laboratories who are working on these
topics since long will make the efforts
more easy ,accurate ,providing the results in shorter time frame .
SUMMARY : The symptoms in hepatic schistosomiasis are vague hence laboratory
diagnosis is carried out for confirmation of the disease. While deciding to
employ diagnostic methodology, it is important to consider our aim of
diagnosing the infection. When our sole aim is treating a sick animal, visiting
a Veterinary Hospital ,coprological diagnosis is recommended but using more
sensitive methods like acid-ether and hatching methods. During post-mortem
,either for collecting blood –flukes or for confirming the infection, perfusion
method is the choice having highest sensitivity . In deciding zoonotic
potentials of a given schistosome species or for surveillance of the infection both parasitological and immunological methods
should be followed as both will supplement each other. As antibody detection
method does not differentiate between present and past infection with
additional disadvantage of invasiveness, antigen detection methods in host’s
excreta are gaining priorities .But it must be remembered that so far all immunodiagnostic
methods are efficient only to identify the infection up-to genus level ( Schistosoma ) and cannot differentiate the infection up-to species levels . Therefore, it is
essential to undertake molecular studies
, if you wish to decide existence of schistosome species, sub-species or
hybrids , in a given geographical area, as no immuno-diagnostic method can reveal such
complexities of schistosomiasis .
REFERENCES :
Agrawal, M.C. 1999. Schistosomosis : an underestimated
problem in animals in South Asia. World Animal Review 92 : 55-57.
Agrawal, M.C. 2000. Final report on National Fellow project
“Studies on strain identification,epidemiology,diagnosis,chemotherapy and
zoonotic potentials of Indian schistosomes. ICAR, New Delhi
Agrawal,M.C. 2005. Present status of schistosomosis in
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Agrawal MC. 2012. Schistosomes and schistosomiasis in South
Asia. Springer India Pvt Ltd , New Delhi
Banerjee PS and Agrawal MC. 1991. Prevalence of Schistosoma nasale Rao 1933 at
Jabalpur. Indian J Anim Sci 61 : 789-791
Chandler AC. 1926. A new schistosome infection of man with note
on other human fluke infections in India. Indian J Med Res 14 : 179-183
Gadgil RK and Shah SN. 1952 . Human schistosomiasis in India. J Med Sci 6 :
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Sumanth S ,D’Souza PE and
Jagannath MS. 2003. Immunodiagnosis of nasal and visceral
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