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.