Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
FINAL REPORT ON
“STUDY OF ZOONOTIC WATERBORNE PROTOZOAN PARASITES FROM FRESHWATER BODIES IN SOLAPUR, MAHARASHTRA STATE, INDIA.”
UNDER MINOR RESEARCH PROJECT (WRO) PUNE
SUBMITTED TO UNIVERSITY GRANTS COMMISSION, WRO, PUNE
SUBMITTED BY, DR. DAMA LAXMIKANT BASAVRAJ M.SC., Ph.D., MSFT, FZSI, FIAAB, FSLSC, FSAB (PRINCIPAL INVESTIGATOR) HEAD AND ASSISTANT PROFESSOR DEPARTMENT OF ZOOLOGY, D.B.F. DAYANAND COLLEGE OF ARTS AND SCIENCE, SOLAPUR (M.S.) INDIA. REACCREDITED BY NAAC WITH „A‟ GRADE RECOGNIZED BY UGC AS „COLLEGE WITH POTENTIAL FOR EXCELLENCE‟ (AFFILIATED TO SOLAPUR UNIVERSITY, SOLAPUR)
AND
DR. MUSHAN LAXMI CHANDRAKANT, M.SC., Ph.D. (CO- INVESTIGATOR) ASSISTANT PROFESSOR, DEPARTMENT OF ZOOLOGY, D.B.F. DAYANAND COLLEGE OF ARTS AND SCIENCE, SOLAPUR (M.S.) INDIA.
OCTOBER, 2015
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
INDEX
Sr.No. Title Page No.
i Acknowledgement 1
ii Final Report 2
1 Introduction 6
2 objectives 17
3 Review of literature 18
4 Materials and methods 19
5 Results and discussion 25
6 Conclusion 43
References 47
Publications 50
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Acknowledgement
I extend my deepest sense of gratitude and express sincere thanks to Shri Mahesh Chopraji, (Local Secretary, Dayanand Institutions, Solapur and Dr. S.K. Vadagbalkar, Principal, D.B.F. Dayanand College of Arts and Science, Solapur for providing laboratory facility. Principal Investigator and Co-Investigator of this project thank the University Grants Commission (UGC) WRO Pune, (India) for financial assistance provided to carry out this work through minor research project entitled “Study of waterborne Protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.”, (File No. 47- 1487/10 (WRO) Dated: 7th October, 2010).
We also thank to Dr Ramesh Chondekar, Assistant professor, Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, for their suggestions and comments on various drafts of this Project. We also thankful to non-teaching staff of Zoology department and administration office, account department and library for the cooperation extended during my project work. I am very much thankful to all my colleagues, post-graduate, research students and my friends at D.B.F. Dayanand College of Arts and Science, Solapur, for providing much needed stimulus and support for successful completion of project. I have no words to acknowledge my family members for the patience, encouragement and unconditional support bestowed on me all the time. Above all I am grateful to the Almighty with eternal gratitude and humility for his blessings.
Dr Laxmikant Basavraj Dama
Principal Investigator
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Annexure -III
UNIVERSITY GRANTS COMMISSION
BAHADUR SHAH ZAFAR MARG NEW DELHI – 110 002.
Annual/Final Report of the work done on the Minor Research Project.
(Report to be submitted within 6 weeks after completion of each year)
1. Project report No. 1 st /2 nd /3 rd /Final FINAL
2. UGC Reference No. Ref. File No. 47-1487/10 (WRO) Dated: 7th October, 2010
3. Period of report: from 11-11-2011 to 10-11-2012
4. Title of research project: Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
5. (a) Name of the Principal Investigator Dr Laxmikant Basavraj Dama
(b) Deptt. and University/College where work has progressed Zoology Department
D.B.F. Dayanand College of Arts and Science, Solapur (M.S.), India.
6. Effective date of starting of the project 11-11-2010
7. Grant approved and expenditure incurred during the period of the report: a. Total amount approved Rs. 1,01000=00 (One Lakh One Thousand only) b. Total expenditure Rs. Rs. 1,02448=00 c. Report of the work done: (Please attach a separate sheet) Final Bound copy Attached. i. Brief objective of the project
-Survey of different water bodies in Solapur.
-To study the contaminated water for protozoan infection and taxonomic identification of free living protozoa.
-Preparation of checklist of protozoa from this region with taxonomic details.
-To study the survey and prevalence of waterborne protozoan pathogens, risks of secondary spread agents and their geographical and socioeconomic factors.
-To minimize the entry of animal wastes to source waters, by controlling animal movements, proper storage and disposal of farm animal wastes, using procedures that
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. will minimize the survival of zoonotic protozoan pathogens, and limiting transport of these wastes in surface water runoff.
-Analytical epidemiological study for waterborne protozoan diseases and to provide a benchmark for risk assessment modeling and calculations.
-Investigation of waterborne disease outbreaks to guide research as well as evaluate controls and regulations for drinking-water and recreational water.
-Suggestions for prevention and control of waterborne protozoan zoonoses.
-To develop strategies for water source protection and treatment, and food production, reducing the disease burden and increasing economic wellbeing.
-To conduct an intensive health education campaign in the area and establishing new protocols. ii. Work done so far and results achieved and publications, if any, resulting from the work (Give details of the papers and names of the journals in which it has been published or accepted for publication
Three papers are published in International Journal as follows:
1. Jagtap M.N., Dama L.B., Shaik K.A. and Shaik H.G. (2012). Isolation of Phytoplankton’s from three Urban Lakes of Solapur City, Maharashtra State, India- DAV International Journal of Science 1(2): 68-71. 2. Shaikh Afreen S M, Shaikh J. D., Dama L.B., Prashant Bangale and Mohammed Ehtesham (2012). Study of aquatic protozoan in Neher-E-Ambary in Aurangabad vicinity. Trends in Parasitology Research. 1(2): 39-45. 3. Dama S. B. and Dama L.B. (2013). Prevalence Waterborne Entamoeba Histolytica from Freshwater Bodies in Solapur, Maharashtra State, India. Trends in Parasitology Research. 2(1): 22-23. iii. Has the progress been according to original plan of work and towards achieving the objective. if not, state reasons
Yes Progress achieving as per the Objectives iv. Please indicate the difficulties, if any, experienced in implementing the project__Nil______
v. If project has not been completed, please indicate the approximate time by which it is likely to be completed. A summary of the work done for the period (Annual basis) may please be sent to the Commission on a separate sheet – Project Completed
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. vi. If the project has been completed, please enclose a summary of the findings of the study. Two bound copies of the final report of work done may also be sent to the Commission Attached Bound Copy of the Final Report of work done. vii. Any other information which would help in evaluation of work done on the project. At the completion of the project, the first report should indicate the output, such as (a) Manpower trained (b) Ph. D. awarded (c) Publication of results (d) other impact, if any
This project helps our departmental Ph.D. Student Shri Talbhandare Siddharth.
Three papers are published; it helps for researchers in the field of Parasitology, Zoology and Environmentalists. Two awareness training programmes are conducted under this project to create awareness of cloth washers besides the lake.
SIGNATURE OF THE PRINCIPAL INVESTIGATOR
Dr Dama Laxmikant Basavraj Dama
SIGNATURE OF THE COINVESTIGATOR
Dr Mushan Laxmi Chandrakant
REGISTRAR / PRINCIPAL
Dr Vadagbalkar S.K.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
INTRODUCTION
Protozoa are unicellular microscopic eukaryotes that have a relatively complex internal structure and carry out complex metabolic activities. Some protozoa have structure for propulsion or other types of movement. The protozoa are considered to be a sub kingdom of the kingdom Protista, although in the classical system they were placed in the kingdom Animalia. More than 65,000 species have been described, most of which are free living organisms. Protozoa are found in almost every possible habitat. Anton van Leeuweenhoek was the first person to see protozoa, using microscopes he constructed with simple lenses. Protozoa are among the most fascinating organisms that can be studied in the laboratory. The Protista Kingdom has 7 groups that are divided into fifteen Phyla, these subdivisions show the wide range of morphology and function that demonstrate the basic properties of living matter. The different phyla are distinguished from one another by such features as structure, means of locomotion, and formation of spores, although the locomotory organelles are the primary distinguishing feature. There are three main locomotory organelles found in the different classes of protozoa, and they are pseudopodia, cilia, and flagella.
Protozoa is one of the zoonotic agents and it can be transmitted from animals to humans either directly or indirectly. Indirect transmission means that the agents are passed from animals to humans via food, water, environment, vectors, etc. Waterborne zoonotic agents include bacteria, protozoa, viruses, and helminthes, but bacteria and protozoa are the zoonotic agents that are most often implicated in waterborne disease
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. outbreaks.These diseases are gastrointestinal tract infections, except for leptospirosis and toxoplasmosis, which are systemic infections. Therefore, diarrhea is the common symptom of these diseases except for leptospirosis and toxoplasmosis. Wild cases of bacterial gastrointestinal infections are self-limiting, and antibiotic treatment is not recommended, especially for enterohaemorrhagic E. coli infection. Most of the diseases are effectively curable. The severity of the symptoms depends on the infective organisms as well as the health status of the infected person. Most of the diseases give rise to health consequences.
Protozoan pathogens, including microsporidia, amoebae, ciliates, flagellates, and apicomplexans, originating in human or animal feces have been found in surface waters worldwide. Many have been found infrequently or in low numbers or have been identified only by general morphological features that are not precise. The zoonotic protozoa that are emerging or are of renewed interest because their spread is associated with water and include several species of microsporidia, the amoeba
Entamoeba histolytica, Balantidium coli Giardia spp. The ability to conduct epidemiological studies relating these organisms to human infections, animal sources, and water will now provide a basis for planning, prevention and control strategies.
In Maharashtra following researchers have observed the waterborne protozoa in freshwater by Gupta (2011), Giardia, Cyclospora, Balantidium coli, and human infective microsporidia (e.g., Encephalito zoonintestinalis, Encephalit zoonhellem, Encephalito
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. zooncuniculi, and Enterocyto zoonbieneusi) are human enteric parasites in which transmission is associated with water (Wolfe, 1992; Ortega et al., 1993).
Although Giardia lamblia (syn. G. intestinalis, G. duodenalis) and
Cyclosporacayetanensis cause serious diarrheal illness in adults and children worldwide the infections usually respond well to pharmacological treatment (Wolfe,
1992; Ortega et al., 1993; Mansfield and Gajadhar, 2004). Human-infective microsporidia cause serious illness in immunodeficient patients; however, they also respond well to pharmacological treatment. Cryptosporidium parvum, G. lamblia, and human-infective microsporidia are anthropozoonotic pathogens. There are five critical elements in the transmission of zoonotic Protozoans throughwater:
1. Source of the infectious agent
2. Specific water-related modes of transmission
3. Attributes of the organism that allow it to survive and possibly multiply and to move into and within the aquatic environment.
4. Infectious dose and virulence factors of the organism.
5. Host susceptibility factors.
Zoonotic parasites may be separated into four categories such as a) Direct zoonotic parasites infecting humans directly from animals e.g. E. histolytica, C. parvum, T. gondii b) Meta-zoonotic parasites infecting humans from invertebrate intermediate hosts e.g.
Fasciola gigantica, Diphyllobothrium latum, Dipylidium caninum, Dirofilaria immitis,
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. c) Cyclo-zoonotic parasites having vertebrate intermediate hosts e.g.Taenia saginata,
Tania solium, Echinococcus granulosusetc. d) Sapro-zoonotic parasites meaning that parasites can infect humans from soil or water e.g. Ancylostoma caninum, Ascaris suum, Strongyloides stercoralisetc (Samad,
2011).
Solapur District
Solapur district, Maharashtra State, India (Figure-1), is bounded by 17º05' North latitudes to 18º 32' North latitudes and 74º 42' East of 76º 15' East longitudes
Figure 1. Map of Solapur shows in blue colour.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Total geographical area of Solapur district is 14895² K.m. divided into eleven tahsils.
The Population is 32.4 lakhs in eleven tahsils of District. It is bounded from the North by
Osmanabad district and Ahmednagar district, on North-East by Satara district and at the
South & East it has common boundary of Karanataka state. The temperature is high in summer season. Rainfall varies from East to West between ranges of 200 to 600 millimeters. Solapur is an important city in Maharashtra most well-known for its textile industry, and bed sheets in particular.
LAKES IN SOLAPUR CITY
In Solapur City three lakes are present namely Sambahji Lake, Shiddeshwar Lake and Ekruk Lake. The Lake latitude are shown in Table 1.
Table 1. Lake Latitude / Longitude of Lakes in Solapur
Site N Latitude E Longitude
Shiddeshwar lake 1740.431 7554.271
Sambhaji lake 1738.370 7554.370
Ekruk Lake (Hipparga lake) 1744.784 7554.982
Sambhaji Lake
The original name of Sambhaji Lake (Kambar Talav) was QAMAR lake. This was named after daughter’s name of Aurangzeb-the Mughal Emperor. But now this is known as Kambar Talav. It is also known as Sambhaji Lake (Figure 2). The Sambhaji Lake is a well-known attraction in the city where you will see locals flocking in the evening and weekends for a fresh breath of air or just a long walk along the banks.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure-2. Sambhaji lake, Solapur, Maharashtra State, India(Source Google Map)
Shiddeshwar laker
Siddheshwar Temple is the temple of “Lord Shiddheswara”. The temple, situated in the middle of the lake, is picturesque; with a backdrop of the fort looming behind it. The temple was built by a yogi, Shri Siddharameshwar, who was a devotee of Sri
Mallikarjuna of Srisailam. He was on his way to Srisailam, when he was ordered by his guru to return to Solapur and many Shiva lingams. He duly returned to Solapur and began his work, starting from this temple. In all, he installed 68 Shiva lingams in
Solapur, Another feature of this temple is the garden that has a shrine dedicated to
Saint Nalatwad. Other shrines in the temple complex are dedicated to Lord Vithoba and
Goddess Rukmini. Around the temple Siddheswar lake (Figure 2) situated.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
A
B
Figure- 3. Shiddeshwar lake Solapur, Maharashtra State, India. (Source Google Map) A. Shiddheswar temple B. Over view by satellite image (Google map)
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Ekruk lake
The Ekruk lake (Figure-4), the largest artificial lake in the Bombay Presidency, lies five miles north-east of Solapur. The scheme was prepared in 1863 and sanctioned in 1866.
It comprises a reservoir formed by an earthen dam 7,200 feet long and seventy-two feet in greatest height and three canals. The dam is thrown across the valley of the Adhila, a feeder of the Sina, which has a drainage area of 160 square miles above the lake. The lake is sixty feet deep when full, and holds 3,350 millions of cubic feet. The area of water surface is 4,640 acres or 7 square miles. Of the canals one on each bank is at a high level, designed for four months' watering, and the third on the left bank is at a low level, designed for a twelve months discharge. Of the two high level canals the right bank canal is eighteen miles long, discharges sixty cubic feet a second, and commands
565 acres; and the left bank canal is four miles long, discharges twenty-five cubic feet a second, and commands 856 acres. The low level left bank canal is twenty-six miles long, discharges seventy cubic feet a second, and commands 10,601 gross acres. The canals are bridged and regulated throughout and can be lengthened so as to command a larger area. The low level canal flows close past the town of Solapur. The work was begun in 1866, and the dam was closed in December 1869. Some water was supplied to the kharif or rain crop of 1871-72. At the end of 1876-77 the work was completed, except the masonry heads to distributaries and the last two miles of the low level canals and the last twelve miles of the high level right bank canal. By the end of 1881-82 all the works connected with the Ekruk lake were completed at a total cost of about 121,262
(Rs. 12,12,620). In 1882-83, of 15,320 acres, the arable area under command, 1,306
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. acres were watered and paid 524 (Rs. 5,240) for water rates. Besides tillage water, the
Ekruk Lake supplies drinking water to the town of Solapur.
Figure-4.Ekrukh Lake (Hippargalake), Solapur, Maharashtra State, India. (Source
Google Map)
Water born protozoan causes following diseases namely
1. Amoebiasis- E. histolytica, E. coli
2. Balantidiosis- Balantedium coli
3. Giardia
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Amoebiasis
Amoebiasis is a parasitic protozoan disease caused by the protozoan Entamoeba histolytica. This disease is prevalent worldwide but is most prevalent and severe in tropical countries where prevalence rate may exceed 40% under conditions of crowding, poor sanitation and poor nutrition. Amoebiasis is the third leading cause of death from parasitic diseases worldwide, with its greatest impact on the people of developing countries. WHO estimates that approximately 50 million people worldwide suffer from invasive amoebic infection annually, resulting in 40,000 to 100,000 deaths annually (Haque, 2007). Transmission is usually by ingestion of infective cysts which contaminated with food and water. The cysts remain viable for 12 days in moist environment and 30 days in water. It is characterized in humans by mild to recurrent diarrhea, stools streaked with blood and mucus. Hepatic amoebiasis is characterized by fever, hepatomegaly, moderate colitis, pain and localized tenderness. Amoebiasis disease can be prevented by strict sanitation and personal hygiene, protective clothing and gloves. In addition, fecal contamination of feed and water should be prevented, and fecal screening and adequate cooking to destroy the cysts need to be done.
Balantidiosis
This disease is found worldwide with more prevalence in the tropics. Balantidiasis is an infectious feco-oral transmitted zoonotic disease caused by a single-celled protozoan called Balantidium coli that infect the digestive system. This protozoa infect a wide variety of mammals and especially common in monkeys and pigs which generally regarded as the main reservoir for human infection. Prevalence in pigs ranges from 20 to 100% and human Balantidiosis usually exhibits an increased prevalence in
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. communities that like in close association with pigs. It is transmitted primary by eating food or drinking water that has been contaminated by human or animal feces containing
B. coli cysts. Balantidiosis associated with diarrhea in cattle and water buffaloes (Islam et al., 2000). Human-to-human transmission has also been documented and this method of transmission is likely to occur in environment with crowding and poor personal hygiene.
Giardiasis
Giardiasis is an intestinal illness caused by a microscopic parasite called Giardia lamblia. It is a fairly common cause of diarrheal illness. Most people become infected with giardiasis by drinking water contaminated with the Giardia parasite, or through direct contact with an infected person. The giardiasis infection can also be passed on if an infected person doesn't wash their hands properly after using the toilet, then handles food eaten by others. Food can also be contaminated if it is washed with infected water.
Giardiasis occurs almost everywhere in the world, but is particularly widespread where access to clean water is limited and sanitation is poor. It can affect people of all ages but is most common in young children and their parents. This is because things like nappy changing increase the risk of infection. Some people can carry giardia parasites without experiencing any symptoms. Symptoms of giardiasis generally show up one or two weeks after exposure. Common symptoms include: fatigue, nausea, diarrhea or greasy stools, loss of appetite, vomiting, bloating and abdominal cramps, weight loss, excessive gas, headaches and abdominal pain.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
OBJECTIVES
Survey of different water bodies in Solapur.
To study the contaminated water for protozoan infection and taxonomic
identification of free living protozoa.
Preparation of checklist of protozoa from this region with taxonomic details.
To study the survey and prevalence of waterborne protozoan pathogens, risks of
secondary spread agents and their geographical and socioeconomic factors.
To minimize the entry of animal wastes to source waters, by controlling animal
movements, proper storage and disposal of farm animal wastes, using
procedures that will minimize the survival of zoonotic protozoan pathogens, and
limiting transport of these wastes in surface water runoff.
Analytical epidemiological study for waterborne protozoan diseases and to
provide a benchmark for risk assessment modeling and calculations.
Investigation of waterborne disease outbreaks to guide research as well as
evaluate controls and regulations for drinking-water and recreational water.
Suggestions for prevention and control of waterborne protozoan zoonosis.
To develop strategies for water source protection and treatment, and food
production, reducing the disease burden and increasing economic wellbeing.
To conduct an intensive health education campaign in the area and establishing
new protocols.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
REVIEW OF LITERATURE
Waterborne diseases are a major cause of mortality worldwide. The association between warmer temperatures and the occurrence of disease suggests that rates of waterborne illnesses are likely to increase with rising temperatures (Greer et al., 2008).
The temperature of Solapur is very high in summer (40-450C). The close relationship between climate, environment and infectious diseases in the developing world is well recognized. In 2007, Haque observed that that approximately 50 million people worldwide suffer from invasive amoebic infection annually, resulting in 40,000 to
100,000 deaths annually. In India , Bhandari et al. (2001) studied the role of protozoa as risk factors for persistent diarrhea. In 2011, Singh et al. observed climate change,
Zoonosis in India. Umakant Kambale (2012) studied free living protozoan from Pawai
Lake, Mumbai (Maharashtra). Alam et al., (1979). Observed the Blantidiasis in cattle.
Protozoan infections are generally spread by the classic fecal–oral route. Infection results from ingestion of cysts in water or food; trophic organisms cannot survive passage through the stomach, except in cases of low stomach acidity (Schuster et al.,
2004). E. histolytica is responsible for amebic dysentery (Albach and Booden, 1978;
Haque et al., 2003; Ravdin, 1986). Once reaching the colon, the invasive amebae attack the intestinal epithelial surface, causing flask-shaped ulcerations in the wall and producing a blood- and mucus-flecked loose stool. Amebae can also disseminate from the intestinal tract via the hepatic portal system to form discrete abscesses in the liver, lungs, or brain. In severe amebiasis cases, the intestinal wall can perforate releasing amebae into the peritoneal cavity. Water is one of the very crucial natural resources.
About 97% of the water on the earth is in the oceans. The ocean water is salty and is
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. not suitable for human consumption. The remaining 3% of the total water is available in the form of ice sheets, cloud, river, lakes, ponds, well and springs. Only 1-1.6% of the
3% of water is useful for human consumption (Gulani, 2007). The disposal of agricultural waste and untreated sewage into water bodies adversely affect the plant and animal life. About 65,000 species of protozoan parasites are in the World. The most common zoonotic protozoan diseases are Amoebiasis, Giardiasis, Balantidiasis,
Cryptosporidiosis and Toxoplasmosis (Hoque et al., 1994). The present study includes to study the zoonotic waterborne protozoan parasites from freshwater bodies in
Solapur, Maharashtra State, India.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
MATERIALS AND METHODS
Methodology
During February 2011 to January 2012 (summer and monsoon winter), estimation of different physic-chemical parameter of the water samples are done as per standard method for the examination of water and microscopic observation on free living ciliates were done after their movements were slowed down with 10% methyl cellulose.
Field operations
The study is conduct in different water bodies in Solapur city shown in Figure 5, 6 and 7 including study spots).
Sample Preparation
Surface water samples were collected from identified stations of the lake at a depth of one feet using polythene cans of two liter capacity for a period of one year, from
February 2011 to January 2012 at monthly intervals. Transparency and Vertical attenuation coefficient (VAC) was determined by sacchi disc. pH was measured by using pH meter. The chemical analysis was carried out following the methods suggested by Trivedy and Goel (1986) and APHA (1995). For the enumeration of protozoa, each site in a 50mL sample bottle by filtering about 50 liters of water through plankton net. Sample was fixed simultaneously with 20 ml of 1% lugol solution for sedimentation. This sedimented sample was observed under microscope. The identification of Protozoa up to the level of genus was made with the help of literature available. Although considerable work has been done on the limnological studies on some lakes of Maharashtra. The three lakes of Solapur city selected for the present study remains scientifically unexplored.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Sampling location and station
Spot 1 (South part- Sambhaji Lake)
Spot 1 (East part- Sambhaji Lake)
Figure- 5.Sambhajilake, Solapur, Maharashtra State, India Spot 1 and 2
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Standard protocol use for the description of fixation, storage, staining and mounting techniques commonly used to prepare the material for study.
Spot 1 (North part- Shiddheswar Lake)
Spot 1 (South part- Shiddheswar Lake)
Figure- 6. Shiddeshwar lake Solapur, Maharashtra State, India. Spot 1 and 2
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Spot 1.(South part- Ekrukh Lake)
Spot 2 (North part- Ekrukh Lake)
Figure- 7.Ekrukh Lake (Hippargalake), Solapur, Maharashtra State, India. Spot 1 and 2
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Laboratory procedures for Protozoan parasites: Standard collection, Staining, preservation for the proposed work. The following stains are use for protozoa staining namely, Romanowsky Stains, Giemsa stain, Modified Ziehl-Neelsen stain, Phenol- auramine stain, Trichrome Stain, Funguqual (Uvitex 2B or Calcofluor) stain, Iron
Haematoxylin stain. The routine analysis of protozoan parasites in water samples relies upon direct microscopic detection after concentration of particulate matter by filtration or centrifugation.
Identification
A list of some of the major taxonomic works useful for identification of specimens to the generic level. This is intended as a starting point, not a definitive listing of taxonomic works. Microscopic examination of and recording of the results by using statistical methods.
Taxonomic Aids and Keys to Species
Identification of many parasite species may require consultation of original descriptions in the primary literature. However, for most common groups, identification to genus and often to species can be done through the synthetic keys. Voucher specimens are deposited in the permanent collection of a recognized museum, for future reference and use for researchers.
Statistical analysis
Detection of prevalence, abundance and frequency is measure by Standard statistical analysis methods.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
RESULTS AND DISCUSSION
The present research work covers physico-chemical factors, prevalence of zoonotic waterborne protozoa from water bodies of Solapur city. During the study total number of
3 Genus and with 4 species has been recorded namely E. histolytica, E. coli, B. coli and
Giardia spp.
A)Entamoeba histolytica
The results shown in Table 2 and Figures 8,
Taxonomic Classification
Classification: Entamoeba histolytica
Domain: Eukaryota
Kingdom Protista
Subkingdom Protozoa
Phylum Sarcomastigophora
Subphylum Sarcodina
Class Lobosea
Order Amoebida
Family Endamoebidae
Genus Entamoeba
Species histolytica
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 8: Entamoeba histolytica
Table 2. Percentage of Prevalence of Entamoeba histolytica (%) of zoonotic waterborne Protozoa in Sambhaji Lake during the Period February 2011 to
January 2012.
Sr. Month Sample Tested Positive samples of E. histolytica % of Positive samples No
Sambhaji Shiddheswar Ekrukh Sambhaji Shiddheswar Ekrukh Sambhaji Shiddheswar Ekruk Lake Lake Lake Lake Lake Lake Lake Lake h Lake
1 Feb 10 10 10 5 2 1 50 20 10
2 March 10 10 10 3 2 0 30 20 00
3 April 10 10 10 2 1 0 20 10 00
4 May 10 10 10 0 0 0 00 00 00
5 June 10 10 10 0 0 0 00 00 00
6 July 10 10 10 4 1 2 40 10 20 7 Aug 10 10 10 6 2 3 60 20 30
8 Sept 10 10 10 6 4 3 60 40 30 9 Oct 10 10 10 8 3 2 80 30 20
10 Nov 10 10 10 7 4 4 70 40 40
11 Dec 10 10 10 6 2 2 60 20 20
12 Jan 10 10 10 6 1 1 60 10 10
Total 120 120 120 53 22 18 44.16 18.33 15.00
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 9. Prevalence of percentage of E. histolytica from Sambhaji Lake, Solapur during the period February 2011 to January 2012.
Figure 10. Prevalence of percentage of E. histolytica from Shiddeshwar Lake,
Solapur during the period February 2011 to January 2012.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 11. Prevalence of percentage of E. histolytica from Ekruk Lake, Solapur during the period February 2011 to January 2012.
During the year Feb. 2011 to January 2012 total 360 water samples were collected
(each lake 120 water samples for 3 lakes), of which 53 positive samples of E. histolytica
(Table-2) were found out of 120 samples and the prevalence is 44.16%. In Shiddheswar
Lake 22 positive samples of E. histolytica were found out of 120 samples and the prevalence is 18.33%. In Ekrukh Lake 18 positive samples of E. histolytica (Figure- 8) were found out of 120 samples and the prevalence is 15%. In the month of May and
June there was no protozoa recorded from the sample and hence the prevalence reaches to zero. The maximum percentage of prevalence was recorded in the month of
October to January which gradually decreases up to April (0.0%). In Ekrukh lake.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
B) Entamoeba coli
The results shown in Table 3 and figure 12, 13, 14,15
Classification: Entamoeba coli
Domain: Eukaryota
Kingdom Protista
Subkingdom Protozoa
Phylum Amoebozoa
Subphylum Sarcodina
Class Archamoebae
Order Amoebida
Family Endamoebidae
Genus Entamoeba
Species coli
Figure 12. Entamoeba coli
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Table 3. Percentage of Prevalence of E. coli (%) of zoonotic waterborne Protozoa in Sambhaji Lake during the Period February 2011 to January 2012
Sr. Month Sample Tested Positive samples of E. coli. % of Positive samples No Sr Month Sambhaji Shiddheswar Ekrukh Sambhaji Shiddheswar Ekrukh Sambhaji Shiddheswar Ekrukh No. Lake Lake Lake Lake Lake Lake Lake Lake Lake 1 Feb 10 10 10 3 2 1 30 20 10 2 March 10 10 10 1 1 0 10 10 00 3 April 10 10 10 1 1 1 10 10 10 4 May 10 10 10 0 0 0 00 00 00 5 June 10 10 10 0 0 0 00 00 00 6 July 10 10 10 0 0 0 00 00 00 7 Aug 10 10 10 2 0 0 20 00 00 8 Sept 10 10 10 3 2 1 30 20 10 9 Oct 10 10 10 3 2 2 30 20 20 10 Nov 10 10 10 3 4 2 30 40 20 11 Dec 10 10 10 2 1 3 20 10 30 12 Jan 10 10 10 1 1 2 10 10 20 Total 120 120 120 19 14 12 15.83 11.66 10
During the year Feb. 2011 to January 2012 total 360 water samples were collected
(each lake 120 water samples for 3 lakes), of which 19 positive samples of E. coli were found out of 120 samples and the prevalence is 15.83%. In Shiddheswar Lake 22 positive samples of E. coli were found out of 120 samples and the prevalence is
11.66%.
In Ekrukh Lake 18 positive samples of E. coli were found out of 120 samples and the prevalence is 10%. In the month of May, June, July and August, there was no protozoa recorded from the sample and hence the prevalence reaches to zero. The maximum percentage of prevalence was recorded in the month of September, October and
November.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 13. Prevalence of percentage of E. coli from Sambhaji Lake, Solapur during the period February 2011 to January 2012.
140
120
100 Sample Tested 80 Shiddheswar Lake 60
40 Prevalance %
20
0
July
May
Total
June
April
March
August
January
October
February
December
November September
Figure 14 . Prevalence of percentage of E. coli from Shiddheswar Lake, Solapur during the period February 2011 to January 2012.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 15. Prevalence of percentage of E. coli from Ekrukh Lake, Solapur during the period February 2011 to January 2012.
32
Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
C) Balantidium coli
The results shown in Table 4, Figure 16, 17, 18, 19.
Classification: Balantidium coli
Domain: Eukaryota
Kingdom: Chromalveolata
Superphylum: Alveolata
Phylum: Ciliophora
Class: Litostomatea
Order: Vestibuliferida
Family: Balantiididae
Genus: Balantidium
Species: coli
Figure 16. Balantedium coli
Table 4. Percentage of Prevalence of B. coli (%) of zoonotic waterborne Protozoa in Sambhaji Lake during the Period February 2011 to January 2012
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Sr. Month Sample Tested Positive samples of B. coli % of Positive samples No Sambhaji Shiddheswar Ekrukh Sambhaji Shiddheswar Ekrukh Sambhaji Shiddheswar Ekrukh Lake Lake Lake Lake Lake Lake Lake Lake Lake
1 Feb 10 10 10 1 1 1 10 10 10 2 March 10 10 10 1 0 0 00 00 00 3 April 10 10 10 0 0 0 00 00 00 4 May 10 10 10 0 0 0 00 00 00 5 June 10 10 10 0 0 0 00 00 00 6 July 10 10 10 0 0 0 00 00 00 7 Aug 10 10 10 0 0 0 10 00 00 8 Sept 10 10 10 0 0 0 00 10 00 9 Oct 10 10 10 0 0 0 00 00 10 10 Nov 10 10 10 0 0 0 00 00 10 11 Dec 10 10 10 1 1 1 10 00 00 12 Jan 10 10 10 2 0 1 20 00 00 Total 120 120 120 5 2 3 4.16 1.66 2.5
During the year Feb. 2011 to January 2012 total 360 water samples were collected
(each lake 120 water samples for 3 lakes), of which 5 positive samples of B. coli were found out of 120 samples and the prevalence is 4.16%. In Shiddheswar Lake 2 positive samples of B. coli species were found out of 120 samples and the prevalence is 1.66%.
In Ekrukh Lake 3 positive samples of B. coli were found out of 120 samples and the prevalence is 2.5%. In the month of April to November, there was no protozoa recorded from the sample and hence the prevalence reaches to zero. The maximum percentage of prevalence was recorded in the month of December to March.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
140
120
100
80 Sample tested (Sambhaji 60 Lake ) % of Positive Samples 40
20
0
Jan
Oct
Feb
Dec
Aug
Nov
July
Sept
May
Total
June
April March
Figure 17. Prevalence of percentage of B. coli from Sambaji Lake, Solapur during the period February 2011 to January 2012.
Figure 18. Prevalence of percentage of B. coli from Siddheswar Lake, Solapur during the period February 2011 to January 2012.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 19. Prevalence of percentage of B. coli from Ekrukh Lake, Solapur during the period February 2011 to January 2012.
36
Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
D) Giardia Species
The results shown in Table 5 and Figure 20,21,22,23.
Classification: Giardia Spp.
Domain: Eukaryota
Kingdom Protista
Subkingdom Protozoa
Phylum Sarcomastigophora
Subphylum Mastigophora
Class Zoomastigophora
Order Diplomonadida
Family Hexamitidae
Genus Giardia
Figure 20. Giardia Species
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Table 5. Percentage of Prevalence of Giardia species (%) of zoonotic waterborne
Protozoa in Sambhaji Lake during the Period February 2011 to January 2012
Sr. Month Sample Tested % of Positive samples No Sambha Shiddheswa Ekruk Sambhaj Shiddheswa Ekruk Sambhaj Shiddheswa Ekruk ji Lake r Lake h Lake i Lake r Lake h Lake i Lake r Lake h Lake
1 February 10 10 10 1 1 0 10 10 00 2 March 10 10 10 1 0 0 10 00 00 3 April 10 10 10 1 0 0 10 00 00 4 May 10 10 10 0 0 0 00 00 00 5 June 10 10 10 0 0 0 00 00 00 6 July 10 10 10 0 0 0 00 00 00 7 August 10 10 10 0 0 0 00 00 00 8 Septemb 10 10 10 0 0 0 00 00 00 er 9 October 10 10 10 0 0 0 00 00 00 10 Novemb 10 10 10 0 0 0 00 00 00 er 11 Decemb 10 10 10 1 0 0 10 00 00 er 12 January 10 10 10 2 0 1 20 00 10 Total 120 120 120 6 1 2 5.0 0.83 1.66
During the year Feb. 2011 to January 2012 total 360 water samples were collected
(each lake 120 water samples for 3 lakes), of which 6 positive samples of Giardia specieswere found out of 120 samples and the prevalence is 5.00%. In Shiddheswar
Lake 1 positive samples of Giardia specieswere found out of 120 samples and the prevalence is 0.83%. In Ekrukh Lake 2 positive samples of Giardia specieswere found out of 120 samples and the prevalence is 1.66%. In the month of May to November, there was no protozoa recorded from the sample and hence the prevalence reaches to zero. The maximum percentage of prevalence was recorded in the month of January and February 2012.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
The organelles of protozoa has functions similar to the organs of higher animals. The plasma membranes enclosing the cytoplasm also cover the projecting locomotory structures such as pseudopodia, cilia and flagella. The ciliates are a group of protozoan’s characterized by the presence of hair like organelles called cilia, which are identical in structure to flagella but typically shorter and present in much larger numbers with a different undulating pattern than flagella. Cilia occur in all members of the group and are variously used in swimming, crawling, attachment, feeding, and sensation.
Their early appearance as living organism, their adaptability to various habitats and their capacity to remain viable in the encysted condition, probably account for the wide distribution of the protozoa throughout the world.
Figure 21. Prevalence of percentage of Giardia Species from Sambhaji Lake,
Solapur during the period February 2011 to January 2012.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Figure 22. Prevalence of percentage of Giardia Species from Shiddeshwar Lake,
Solapur during the period February 2011 to January 2012.
Figure 23. Prevalence of percentage of Giardia Species from Ekrukh Lake,
Solapur during the period February 2011 to January 2012.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Seasonal physicochemical analysis
The results of Seasonal physicochemical analysis was shown in Table 6 and 7.
Table 6. Seasonal physicochemical analysis of Water of three lakes (February
2011 to January, 2012).
Parameter \ Sambhaji Lake Shiddeshwar lake Ekruk Lake (Hipparga lake) Site Summer Monsoon Winter Summer Monsoon Winter Summer Monsoon Winter
pH 6.3 7.6 8.0 6.4 7.8 7.8 6.6 7.4 7.9 Total Alkalinity 121 110 118 130 124 121 122 109 116
Hardness 312 290 298 334 298 305 175 166 171 (mg/lt) Transparency 24.18 21.0 22.4 21.33 19.9 20.4 18.19 17.1 17.8 (cm) Air temp M 40-41 35-39 32-36 39-41 35-39 32-35 36-40 35-38 30-35 Water temp 28 26 23 26 24 23 27 24 23 Dissolved 7.01 8.2 9.4 7.10 8,4 9.1 9.35 9.9 10.2 oxygen Odour Unpleasant Not found fishy fishy Not Not Not Not Not found found found found found
Colour Blue green Blue Blue green green green Blue Blue Blue green green
All readings are the Mean of Spot no 1 and 2 of three lakes
Where scarce number and poor in forms of protozoans not considered in the present study. From above result it is concluded that the Sambhaji Lake is more polluted than
Shiddeshwar and Ekruk Lake because the cloths were washed by washer man, and sewage water is directly released by the residential societies and other anthroporogenic activities are going on. In geographic areas of high prevalence, well-tolerated infections are often not treated to eradicate the parasite because eradication would lower the individual’s immunity to the parasite and result in a high likelihood of re-infection. High mortality rate of diarrheal cases has been found related to sanitation, unsafe water and hygiene (WHO, 2012). The collected data compared with the observation of Bhosale et al., (2010), Dhimdhime et al., (2012), Chakraborty et al, (1977), Govind (1963), Shashi
41
Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. et al., (2008), Smitha (2007). Our findings are resembles with above observed data.
Faecal contamination of water in extreme events (droughts, floods) could result in increased prevalence of diseases such as echinococcosis, taeniasis, and toxoplasmosis (Singh et al., 2010). Waterborne protozoa caused illnesses are likely to increase with rising temperatures (Greer et al., 2008). The temperature of Solapur is very high in summer (40-450C). The close relationship between climate, environment and infectious diseases in the developing world is well recognized.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
CONCLUSION
The objectives of the project were achieved. The findings of this study suggest that recreational lakes represent a major risk of contamination of protozoan parasites. A protozoan parasite was an organism that lives at the expense of and caused damage to its host, and which cannot survive for long in the absence of the host. Due to the possible health risk, thorough inspection is recommended to be done on recreational lakes, as well as other sources of water. In light of this issue, Solapur citizens and respective authorities such as local city councils needto improve water safety by maintaining the quality of recreational lakes such as routine cleaning up, appropriatebiological reviving methods (i.e. throwing mudballs with micro-organisms) and, most importantly, campaigning awareness programmes to educate public in general and younggeneration in particular. Based on this study, these recommendations are given to overcome the limitations of the study, which are as follows: Confirmation on viability of these parasites could ascertain accurate risk assessment and further prove its relationship with physico-chemical parameters.
Amoebiasis and other studied protozoan diseases can be prevented by strict sanitation and personal hygiene, protective clothing and gloves. In addition, fecal contamination of feed and water should be prevented, and fecal screening of NHP and adequate cooking to destroy the cysts need to be done. This research can be useful for all the discipline of
Protozoology and Life Sciences. lt is in fact convergence of technology and education.
This research project has profound relevance for interdisciplinary teaching and learning and research. Epidemiological study and surveillance activities can provide information
43
Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. about thewaterborne risks of zoonotic agents and assist public health officials in developing control measures to reduce these risks.
This research project will make significant contribution the international knowledge onProtozoologyThese data was helps officials to develop control programmes to improve the quality ofsurface water sources and have called attention to the need for improved protection ofgroundwater sources and distribution systems from contamination. With increasing population migration, ease of international travel, and movement of foodproducts from country to country, international surveillance becomes more important to alert officials about outbreaks in travelers, impending epidemics, and emerging zoonotic pathogens. The statistics may warn of potential animal husbandry, drinking water protection andtreatment, or food production and preparation practices that should be avoided. Closecollaboration is important to ensure that international surveillance systems have acommon basis for comparison purposes and that the information is readily shared.
Changing global disease situation is very important, as climate change increases the risk of waterborne zoonoses by expanding the host, reservoir, and vector base. The provision of safe food and water and the control of zoonoses and associated outbreaks are important challenges to be faced. Public health infrastructure for food and water safety, disease surveillance, control of vectors and disease reservoirs, and public health outbreak response needs to be strengthened. National, Multinational and collaborative scientific efforts to minimize the waterborne protozoans and spread of these diseases is
44
Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. to be controlled. As per the objective the present study surveyed of different water bodies and their contamination by waterborne protozoans in Solapur. We aware the peoples to minimize the entry of animal wastes to source waters, by controlling animal movements, proper storage and disposal of farm animal wastes, using procedures that will minimize the survival of zoonotic protozoan pathogens, and limiting transport of these wastes in surface water runoff. But these water bodies only one water body
(Ekrukh Lake) was useful for drinking-water. Arrangement of two health education campaign in the research area for students and local peoples. National, surveillance activities can help officials detect outbreaks or epidemics, identifynew and emerging disease threats, and assess temporal trends. Surveillance may consider laboratory- confirmed, physician- or self-reported disease, or disease symptoms and the collection of other information, such as water quality data or information about disease or infection in animal populations. Because zoonotic agents can be transmitted by contaminated drinking-water, recreational water, or food and by direct or indirect contact with infected humans and animals, epidemiological analyses are needed to evaluate waterborne transmission.
The timely investigation of an outbreak by a multidisciplinary team (e.g., epidemiologist, engineer, water quality specialist) with appropriate laboratory assistance can provide information about the mode of transmission, the etiologic agent, sources of contamination, and deficiencies in water and wastewater treatment technologies and watershed protection programmes. This research project will have tremendous significance on education, in general and in research. As an outcome of this project
45
Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India. systematic check of taxonomic value of waterborne protozoa from this region will be prepared. The dearth of data on the prevalence of these zoonotic protozoa in surface waters is related to the lack of rapid and sensitive methods to recover and detect the encysted stages in the aquatic environment. The present study will provide the updated information of various aspects of waterborne protozoa and their distribution, composition, taxonomic details, symptoms and health consequences. This project will fill the existing lacuna in the knowledge.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
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Shashi T.R., Shekhar B.R. Kiran E.T., Puttaiah Y. Shivaraj and Mahadevan K.M.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
PUBLICATIONS
University Grants Commission (UGC) WRO Pune, (India) for financial assistance provided to carry out this work through minor research project entitled “Study of waterborne Protozoan parasites from freshwater bodies in Solapur, Maharashtra State,
India.”, (File No. 47-1487/10 (WRO) Dated: 7th October, 2010). Under this project we publish three Research papers in International Journals as follows.
4. Jagtap M.N., Dama L.B., Shaik K.A. and Shaik H.G. (2012). Isolation of Phytoplankton’s from three Urban Lakes of Solapur City, Maharashtra State, India- DAV International Journal of Science 1(2): 68-71.
5. Shaikh Afreen S M, Shaikh J. D., Dama L.B., Prashant Bangale and Mohammed Ehtesham (2012). Study of aquatic protozoan in Neher-E-Ambary in Aurangabad vicinity. Trends in Parasitology Research. 1(2): 39-45.
6. Dama S. B. and Dama L.B. (2013). Prevalence Waterborne Entamoeba Histolytica from Freshwater Bodies in Solapur, Maharashtra State, India. Trends in Parasitology Research. 2(1): 22-23.
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Study of zoonotic waterborne protozoan parasites from freshwater bodies in Solapur, Maharashtra State, India.
Name and Signature
Principal Investigator
Dr. Dama Laxmikant Basavraj
Head and Assistant Professor, Department of Zoology, D.B.F. Dayanand College of
Arts and Science, Solapur
Co- Investigator
Dr. Mushan Laxmi Chandrakant
Assistant Professor, Department of Zoology, D.B.F. Dayanand College of Arts and
Science, Solapur
Principal
Dr Vadagbalkar S.K.
D.B.F. Dayanand College of Arts and Science, Solapur
51