CONTRIBUTIONS IN THE FIELD OF
PARASITOLOGY
In the professional career, I have searched gaps in the field of
my specialization and tried to fill some of them resulting in original
contributions in parasitology. Following is the summary of the work carried out
by me with my research associates.
STEPHANOFILARIASIS:
Under the able
guidance of late Prof. S.C. Dutt, I worked on ear-sore in buffaloes caused by Stephanofilaria zaheeri, Singh, 1958 for
my M.V.Sc. thesis. Pevalence of ear-sore was in proportion to age of the
buffaloes reaching to 90-100% in old animals. Apparently healthy buffalo skins
also possess adult males. It is futile to conduct skin biopsy for judging drug
efficacy because of a lower filarial concentration even in clinical cases.
In.vitro testing showed malathion to possess anti filarial activity too.
Interestingly, all the cattle examined grossly or histopathologically were non
compatible to S.zaheeri. These findings and our knowledge on stephanofilaria
have been discussed in our review on
Stephnofilarial dermatitis in India (Vet. Res. Comm, 8:93. 1984).
SARCOCYSTOSIS IN INDIA :
We developed a method for detecting developing sarcocysts in
experimentally infected goats (J.Vet. Para, 1, 68, 1987) and a method
for collecting large number of sporocysts from experimentally infected dogs. The
transmission potential of dogs was assessed (Indian J. Anim. Sci. 57,
1101, 1987). The life cycle and
associated pathogenesis of Sarcocystis
capracanis has been studied in India.
Sarcocysts of Sarcocystis spp. were reported from
albino rats from India, which may be tried to develop as a laboratory model for
studying sarcocystosis (5th National Congress Parasitology 1993, Udgir.). I was
invited to deliver a lecture on some problems related to sarcocystosis in India
in the Third Asian Congress of Parasitology at LUCKNOW in 1993.
WILD LIFE PARASITES :
I was asked to
make a survey of wild animals of Kanha National Park, Kanha, Madhya Pradesh for
parasitic infections. The survey revealed presence of Toxocara, Ascaris,
Hookworms, Taenia, amphistomes, and Fasciola in wild animals which included
swamp deer spotted dear elephants, tigers and caniids. Interestingly, the
tigers and a wild cat were excreting Paragonimus
eggs in their faeces beside other eggs. (J. Parasit Appl. Anim. Biol. 3,
115, 1994). A deer-ked bite in man at Kanha was also reported by us (J.
Parasit. Appl. Anim. Biol 3, 129, 1994).Recently we have observed Bivitellobilharzia nairi infection in 8 out of 25 elephants stationed at Kanha
national Park suggesting establishment of the focus in the area which is not a
natural habitat of the elephants (J.Bom Nat His Soc. 97,420,2000).
SCHISTOSOMES AND
SCHISTOSOMIASIS:
Work has been
initiated on schistosomes and schistosomiasis in India by imminent
parasitologists like Montgomery, Rao, Soparker, Gadgil, Srivastava,
Anantaraman, Alwar, Dutt. Indeed, Schistosoma
indicum, S.spindale, S.incognitum, S.nasale, Orientobilharzia.dattai were
discovered and christened in India only. There have been several I.C.A.R.,
I.C.M.R. research projects, presidential addresses in different scientific
congresses on schistosomiasis. Despite these efforts, , there is much to be
done to contain the disease in India for which I have been awarded prestigious
National Fellowship of I.C.A.R., New Delhi. As there was no book on the subject
,I have written a book” Schistosome and schistosomiasis in South Asia” which
has been published in 2012 by German branch of Springer India Pvt Ltd, New
Delhi and marketed globally.
INFECTION AND RECOVERY OF
BLOOD FLUKES:
The percutaneous
method of infecting guinea pigs and rabbits with schistosome cercariae required
proper restraining of the animals and may lead to loss of the cercariae if
proper control is not maintained (Agrawal, 1985 ICMR report). This difficulty
is solved by putting the schistosome cercariae in a polythene bag and fastening
the same either in the paw or tail of the animal (Indian J. Anim. Sci. 68,521
1998) hence may be used in any size of the animal. Earlier, schistosomes were
recovered in India by chopping the organs in saline and searching the flukes
afterwards (see Rao & Ayyar, 1993 down to Biswas, G. 1978). This was not an
accurate method of blood-fluke recovery hence we modified and adapted a
perfusion technique which was quite effective in recovering the flukes from
experimental animals (Agrawal, 1978 Ph.D. Thesis, Indian Vet. J. 56,
682,1979, Indian Journal Parasitology, 5, 227, 1981). This technique was
followed later by other Indian workers (Bhilegaonkar, Das, M., Kaur, N.,
Mishra, A. , Ph.D. theses).
The perfusion technique
has further been improved by replacing automatic pipeting machine with a
vertical water pump (tullu Vijay) commonly used in the desert coolers (Indian
J. Anim. Sci. 68,521,1998). This has not only reduced cost of the
equipment but enabled perfusion of different sized animals effectively.
MAINTENANCE OF FRESH WATER
SNAILS:
A new method of keeping fresh water snails (Lymnaea luteola and Indoplanorbis
exustus )in the polythene bags with addition of mulberry leaves and stored
water at the rate of 50 ml/snail was developed which facilitated easy transport
of the snails ,individual snail observation,no need of daily change of water
and reduced mortality .In another technique ,a cement tank of 8 feet diameter
and 14 inch depth was constructed with an water inlet in the centre and filled
with mud ,water ,aquatic weeds including lotus and wild snails. The tank was
able to sustain life of both the snails with their breeding .The method was
used to infect L.luteola with positive pig faeces and recovering Schistosoma incognitum cercariae in large number after about 40 days .Our method
s will be of great assistance for survey work and to search snail hosts for
specific trematodes. (Indian J Anim Sci. 69,301,1999)
EGG AND GRANULOMA COUNT:
When work on
schistosome immunity was initiated in India, fluke recovery and their size were
the only criteria used for judging immune response (see Tiwari H.C., 1972 to
Shrivastava H.O.P.,1977). Two additional criteria viz. egg and granuloma count
were added by us. These not only judged
effectively the immune response but also assessed tissue damage of the host
(Indian Vet. J, 56, 682, 1979, 61, 451, 1984). My successors
(Bhilegaonkar, 1981, Das, 1984, Kaur, 1985 further confirmed their
dependability in judging immune response and tissue damage of the infected
animals.
HETEROLOGOUS IMMUNITY:
Work on
heterologous immunity was initiated by us in India by preimmunizing the host
with cercariae of S.indicum (Indian Vet. J. 56, 682,
1979) or S.spindale (Indian Vet. J. 60, 321, 1983) in rat and mouse and
challenging subsequently with those of S.incognitum. We established development
of heterologous immunity and factors such as sex of the cercariae, varying
immunizing doses showed no effect while host species, route and number of
exposures did influence immunity (Indian. Vet. J. 61, 451, 1984)
PATENT SCHISTOSOME
INFECTION IN MOUSE:
Our predecessors
failed (Sinha & Srivastava, 1965, Ahluwalia, 1972, Nikhale, 1972) to
demonstrate S.incognitum eggs in
mouse faeces and have postulated a hypothesis for the same. Fresh attempts were
made by us by examining mice faeces but by acid-ether and hatching method. We
proved that mice do pass S.incognitum
eggs in their faeces (Curr. Sci. 54, 640, 1985, Indian J. Med. Res 85,
366, 1986). Thus S.incognitum - mouse
model became an ideal one for chemotherapeutic and immunological studies.
ZOONOTIC POTENTIALS :
It may be recalled that eggs of S.incognitum were first detected in two human stools suggesting
possibility of human schistosomiasis in Indian peninsula by a native
blood-fluke. We have substantiated the possibility of human sufferings by
experimentally infecting rhesus monkeys with S.incognitum and studying liver damage histochemically
histopathologically and biochemically by immature blood flukes. Therefore, man
can not escape the pathology even if he develops the fluke only upto a
particular stage (ICMR Final Report, 1985, Indian J.Med.Res 84,366. 1986,
Indian J. Parasit II, 71, 1987). This has been discussed in our review on Schistosoma incognitum (Helminth Abstr, 58,
239, 1989). Demonstrated widespread cercarial dermatitis in rural population in
India ( J Parasit Dis 24,217,2000 ; Bull.WHO 72,278,2000). Recently reviewed
Present status of human schistosomiasis in India ( Proc. Nat Acad. Sc 75 , 184,
2005)
DIAGNOSIS OF
SCHISTOSOMIASIS:
We have compared
various parasitological diagnostic methods vis a vis serological methods (CHR,
COP, MIT, IHA, RPT) and highlighted ineffectiveness of former methods in
assessing schistosomiasis in our animals ( Indian J. Med. Res 84, 366,
1986, Indian Vet. J. 69, 215, 1992, Indian J. Anim. Sci. 60, 6 28,
1990). The coprological method of examining animals for parasitic infections
has further been simplified by using LDPE polythene bags in place of microslide
and cover slip which has not only reduced the cost of faecal examination but
enabled to dispatch the specimen at any place for seeking experts opinion
(Indian, J. Anim. Sci. 67, 1997). Serological tests like MIT, CHR & ring precipitation were 80-90%
sensitive and specific in diagnosing schistosomiasis (J. Vet. Parasit, 1,
63, 1987 Indian J. Parasit 11, 59, 1987). However, use of J-index showed
change in sensitivity and specificity of
serological tests as per efficacy of the parasitological test employed as
reference test (Indian Vet. J. 68, 1022, 1991). Recent work was done
where mesentery examination was effective not only in detecting schistosomiasis
(raising infection to 80-90% in bovines 48% in goats ) but collecting adult
blood flukes for other work (NATP annual report, 2003). Again, intestinal
scraping proved more simple and effective, in comparison to mesentery
examination but by employing large intestine(NATP annual report, 2003).
CHEMOTHERAPY;
Lithium antimony
thiomalate (Anthiomaline) is the most common drug being used in the field for
treating schistosomiasis albeit relapses
of the cases within 1-3 months of the treatment. Our experimental work in
guinea pigs, infected with S.spindale revealed
the ability of anthiomaline to kill the blood flukes only at the level of
10-39% even with a higher dose ( J Vet Parasit 13,67,1999).Praziquantel , a
more costly drug, could cause only shilf of S.incognitum
flukes to the lungs without killing them significantly (Indian Vet J
69,279,1992).Among refoxanide, CDRI 81/470, oxyclozanide, closantel,
triclabendazole, the last drug was
comparatively more effective in killing S.incognitum
but none could cause a significant blood fluke reduction (Indian Vet
J.76,493,1999,Indian J Anim Sci 69, 97,1999; 70,797,2000, J.Parasit.Dis
23,137,1999) . Field drug trials were under taken against nasal schistosomiasis
in Pull Putta village of Balaghat district using Praziquentel,triclabendazole,rafoxanide
and Anthiomaline. The last drug caused temporary relief with recurrence of the
symptoms ,after two,three months of treatment, Praziquentel was very effective
but treatment was costing Rs 1500/ per animal while triclabendazole was also
effective and was much cheaper than the former drug (chapter 9 of the
book,Agrawal,2012)
LABORATORY MODEL FOR SCHISTOSOMA SPINDALE:
S.spindale is wide spread and causing
great damage to livestock industry. Still it is less studied blood-fluke probably
because of absence of a suitable laboratory model till recent time. Under an
ICMR Project, compatibility of mice, rats, rabbits and guinea-pigs for S.spindale was studied taking parameters
like percentage fluke recovery, sex ratio, egg load, viability of eggs. In
contrast to earlier findings, guinea-pigs proved best suited for maintaining S.spindale in the laboratory (Indian
Vet. J. 73, 131, 1996) followed by rabbits, mice and rats (Indian J.
Anim. Sci. 64, 352, 1994).
CERCARIAL IDENTIFICATION:
We demonstrated
existence of five mammalian schistosome species at Jabalpur (Trans. Roy, Sec.
Trop. Med. Hyg. 85, 231, 1991). However, the greatest impediment in
epidemiological studies in such cases is the difficulty in identifying
schistosome cercariae on morphological grounds alone. On the other hand, animal
inoculation is not only time consuming but also expensive and laborious way of
cercarial identification. Therefore, we examined various methods for
differentiating S.incognitum, S.indicum, S.spindale and O.dattai
cercarial species. We are successful in our endeavour as certain simple
techniques led to identification of cercarial species with considerable
accuracy. Amongst these, mention may be made of a staining technique for furcal
rami (Indian. J. Anim. Sci. 65, 41, 1995) and studies of argentophilic
papillae in the four cercarial species (J. Parasit. Appl. Anim. Biol. 3,
63, 1994).
NASAL SCHISTOSOMIASIS:
Nasal
schistosomiasis or snoring disease was considered to be absent from Jabalpur
since local cattle did not exhibit symptoms of the disease. Our
epizootiological studies supported this view as neither symptoms nor eggs of
the fluke could be observed in the local cattle. Nonetheless, the parasite was
shown to exist in Jabalpur in native buffaloes and cross-bred cattle with the
clinical signs in later cases (Trans. Roy. Soc. Trop. Med. Hyg. 85, 231,
1991, Indian J. Anim. Sci. 61, 789, 1991). We have discussed S.nasale in our review on nasal
schistosomiasis (Helminth. Abstr, 61, 373, 1992).
We have been able
to develop Schistosoma nasale in the
rabbit to adulthood with production of eggs and granuloma in its liver (J.
Parasit. Appl. Anim. Biol. 5, 25, 1996). Thus, a good laboratory model
has been developed which may be used for studying chemotherapy, immunology and
pathology of Schistosoma nasale.
Technologies Developed :-
(1). Infecting large animal with
schistosome cercariae by Precutaneous method.
Infecting
large animals by percutaneous method (Rabbit, Sheep, Goat, Pig, Cattle etc.)
with schistosome cercariae for experimental work on chemotherapy, pathology,
immunology or any other purpose has always been difficult. It required control
of the animals with the use of sedatives and was much time consuming as
individual animal has to be handled else there were chances of spoilage of the
cercariae.
We
have observed that the schistosome cercariae survive in the water with full
biological activities even if kept in a polythene bag. This prompted us to put
counted number of cercariae in polythene bag and tie it in paw of the Guinea
pigs. To our surprise, all the cercariae penetrated the paw skin and the guinea
pigs developed the infection. We repeated this technique by using ear pinna of
the rabbit, tail of the pigs , and ear pinna of the goats. In all these
animals, the schistosome infection developed with recovery of blood flukes
which was not different from that of traditional method (Indian J Anim Sc 68,
521,1998)
(2) Perfusion of animal for recovering
schistosomes:-Recovery of the Schistosomes from
killed animals is important either for confirming diagnosis or assessing
chemotherapy, immunology or any other parameter on schistosomiasis. For
accurate fluke recovery, a perfusion technique is always recommended which
is being carried out by a peristaltic
pump or any other such device.
In
India we have used first time perfusion technique for recovery of the blood-flukes
(Ph.D. thesis 1978 M.C. Agrawal). In the
technique one automatic pipetting machine was used for perfusing the animals.
When national fellow project was undertaken in 1995, it was felt if automatic
pipetting machine or peristaltic pump can be replaced by any other simple
device or equipment. The trials with horizontal water pump commonly used in
desert water coolers proved successful.(Indian J Anim Sc 68,521,1998) Thus the
technique showed two clear cut
advantages over automatic pipetting machine.
(a) While
automatic pipetting machine was useful only in rat & mice, the new
technique with water pump was useful in all the animals of all sizes.
(b) The cost of horizontal water pump is only Rs.
600/- while that of automatic pipetting machine or peristaltic pump is
30,000=00 to 80,000/-.
(3). Use of polythene bag for faecal
examination for parasitic infections :-
All
over the world ,all the medical & veterinary Diagnostic laboratories are
examining faecal samples for parasitic infections by using micro slide &
cover slip. This traditional technique inherits following disadvantages.
(a).
The slide dries up within one hour and it is important to check the sample
within stipulated period.
(b).
The slide cannot be kept as reference material as it spoils within a day or so.
The slide cannot be dispatched outside the city by ordinary mail for taking
second opinion of another expert. The cost of slide & cover slips is more
than Rs. 1.5 /-
We thought to device some simple technique
which may rectify the above problems.
At last we were
successful when processed faecal material was kept in small LDPE polythene bag
whose opening end was sealed with an electric rod. We can observe all the
helminthic eggs as well as protozoan cysts under microscope using 100, 400
magnification. The polythene bags may be stored for about a month and may be
transported to any place by ordinary post. Additionally the cost of the
polythene bags is not more than 25 paise.
(4). Biological Control of fresh water
Snails :- Fresh water snails (Indoplanorbis
exustus, & L.luteola and L. accuminata) are responsible for
transmitting almost all fluke infections in India.Therefore, it is important to
search out any biological control agent for these fresh water snails.
During
our national fellow project we developed a simple technique of keeping fresh
water snails in the artificial pond( Indian J Anim Sc 69,301,1999). The
conditions were simulating to natural conditions. The methodology was quiet
well for 2 years. But after wards we observed diminishing number of snails and
no new snails were coming up. A thorough investigation to the problem for about
a year , revealed that one wingless aquatic insect was responsible for eating
young snails.
The
insects were carnivorous and feeding on snails under natural conditions. These
were identified by Regional Director, Zoological Survey of India Jabalpur as
nymphs of the dragon fly. Our repeated work have shown that these nymphets
breed & develop favorably in the aquatic environment which is also
favourable for the fresh water snails. Since both the organisms are
surviving in the same environment ,it
will be more feasible & appropriate
to develop nymphets as biological control agent for the fresh water snails.
These nymphets are known to feed on mosquito larvae/ pupae ( we have confirmed
the same) and other insects. However, feeding on fresh water snails appears our
original observation. The work may be explored further for control of fluke
infections.
RECENT OPINION AND WORK ON PARASITIC
INFECTIONS :
Though
animal schistosomiasis is a well documented entity in India and South Asia,
there is a controversy over existence of human schistosomiasis in these
countries. Therefore, in recent years , we paid more attention on this subject.
It was surprising to record a high incidence of cercarial dermatitis, caused by
mammalian and avian schistosome cercariae, in rural flock attaining endemic
form in villages e.g. Chattisgarh where a single water pond is being used both
by man and his domestic animals ( Bull World Hlth Org 72, 278,2000 : JPD
24 : 217,2000).
2.Since
coprological methods are least sensitive in diagnosing Indian schistosomiasis,
we applied CHR in human serum with the history of cercarial dermatitis and 20%
cases were confirmed positive by this test .
3.
Recently, urine of the persons, with the history of cercarial dermatitis was
tested for circulating antigens by employing a single step test kit ,developed
in Netherland .Out of 30 cases tested, four turned positive suggesting
excretion of schistosome antigens in the urine.
4,
Our work and papers on schistosomiasis has compelled Government of India,
Health Department to investigate human schistosomiasis in India and have asked
my help in this endeavor.
When
I have taken charge as Dean of the College, I have to interact with many
disciplines and visualized Parasitology
in a holistic perspective.
A
.Here, I recommended Triclabendazole in place of Anthiomaline for treating
schistosomiasis since the former drug has higher efficacy against the infection
along with effect on Fasciola.
B Under integrated farming system, fish
culture is recommended with mixing of animal’s faeces in the ponds while
Parasitologists warn such practices for
chances of increasing fluke diseases. Without putting data (including economics
), it is becoming difficult to convince the scientists of other faculties.
C. For
controlling Parasitic infections, as
Dean ,I am recommending
collecting all the dung from the village etc and converting it into vermi
compost or using in Gobar Gas Plant. There is the need to advocate such
practices and study economy both in terms of Gas/ manure as well as prevention
of common parasitic diseases following such practices.
