pathogenic microbes and endocrine disrupting chemicals...
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PATHOGENIC MICROBES AND ENDOCRINE DISRUPTING CHEMICALS IN WATER AND OREOCHROMIS NILOTICUSIN LAKE VICTORIA, KENYA
Investigator: DR. PATRICK WAWERU KAMUNDIA (BVM, MSC, (UoN))Supervisors
PROF. P. M GICHOHI (BVM, MSc, FRVCS, PhD)PROF. LILLY C. BEBORA (BVM, MSc, PhD)
DR. LUCY W. NJAGI (BVM, MSc, PhD)PROF. PHILIP N. NYAGA (BVM, MPVM, PhD)
INTRODUCTIONThe worlds’ aquatic resources are undergoing continuous pollution
Water in Lake Victoria is contaminated by various pollutants
The microbes include bacteria, viruses and protozoa.
Contaminated water and fish.
Pathogenic bacteria and protozoa -cause infection in man and fish
LITERATURE REVIEWIncreased population - increased effluent loads (Oguttu et al., 2008)
Microbiological and chemical contamination of aquatic environment
Water contaminated - The aquatic products contaminated
Water acts as a media for which bacteria, protozoa and viruses (Davis et al. 2005 ).
Problems to be addressed What pathogenic microbes found in fish and water of Lake Victoria causing disease
in man?
What are the pathogenic microbes causing disease to fish in the Lake Victoria fish?
What are the antimicrobial resistance profiles of the bacterial pathogens?
What are the levels of endocrine disruptors in Lake Victoria?
LITERATURE REVIEW cont. Water-borne bacterial infections: cholera, typhoid fever and bacillary dysentery are wide-spread (Cabral, 2010).
Fish infections: Aeromonas, Streptococcus iniae,
Fish live in the water environment, any contamination of water contaminate the fish
Compromise in public health and food safety (Onyuka et al., 2011).
LITERATURE REVIEW cont. Microbial quality of fish is determined by the quality of water (Fafioye, 2011)
The quality of water is of paramount importance since the pathogens can be transmitted to man causing infections (Wandili, 2011)
Enterobacteriaceae were recovered in fish from Lake Victoria which included Salmonella, E. coli, Proteus, Enterobacter and Shigella (Onyango et al., 2009)
LITERATURE REVIEW cont. Antimicrobial resistance (AMR) and disinfectant resistance - an evolving phenomenon.
Bacteria causing common infections - developed resistance to new antibiotic -worldwide health threat.
Common and affordable disinfectants - rendered ineffective
LITERATURE REVIEW cont. Protozoan enteroparasites : Amoeba, Cryptosporidium and Giardia - major waterborne pathogens.
Single-celled coccidian protozoan
Found in lakes and rivers, when water is contaminated with sewage and animal waste.
Mode of transmission – contaminated water & food (fish).
LITERATURE REVIEW cont. Estrogenic/anti-androgenic compounds include:
the natural estrogen,
17-β estradiol (E2); the synthetic estrogen originating in oral contraceptives, ethynylestradiol (EE2);
Industrial EDC; Bisphenol-A (BPA)
Endocrine Disrupting Chemicals (EDCs) have been reported in sewage and various methods to detect them have been developed.
OBJECTIVES
General objective
To determine pathogenic microbes and endocrine disrupting chemicals in water and Oreochromis niloticus in Lake Victoria, Kenya
The specific objectives of the study:
To determine the prevalence of pathogenic microbes in the lake Victoria water and O. Niloticus,
Determine the antibiotic and disinfectant sensitivity patterns, and disinfectant efficacy of the recovered isolates in water and fish.
Determine and quantify the estrogenic endocrine disruptors in the lake Victoria water
Determine pathological lesions in O. niloticus in lake Victoria
JUSTIFICATIONFish and water are essential components of life in lake Victoria basin
Winam gulf has high population and industrial intensity
Microbial contamination: pathogens lead to infection and therefore ill health
Domestic, sewage & chemical pollution are sources of pathogenic microbes and EDCs which are of public health importance
Need to establish the extent the microbial and endocrine chemicals pollute the water and how it may affect man and fish
Study site Kisumu, in Winam gulf part of Lake Victoria
1. WATER COLLECTION POINTS:
1. Targeted landing sites and
2. rivers and their inlets
2. FISH COLLECTION POINTS:
1. Dunga landing site
2. Tilapia beach landing site
Sample SizesFormula used to determine sample sizes
N = 4PQ / L2 N = [(4 × 0.5 × 0.5) / (0.1) 2] = 384
N =estimated sample size, P = prevalence estimated at 0.5, Q = (1-P), L =precision required (Martin et al., 1987).
Fish for bacteriology = 100 samples x 6 organs = 600 samples
Water for bacteriology= 160 samples
Water for protozoology = 160 samples
Water for EDCs = 90 samples
Fish for histology = 100 samples
Study animal PISCES
Nile tilapia (Oreochromis niloticus)
Herbivorous
Introduced in to the lake in 1960
Economically important, Declining in numbers
SAMPLE COLLECTIONFISH SAMPLES FOR MICROBIAL
MICROBIAL INVESTIGATION
stored in saline and transport media at 4 ̊C until processing at a lab
FISH
SKIN SWAB
1 INCH SQ
GILL SWAB
KIDNEY
5GM
LIVER
5GM
SPLEEN
5GM
STOMACH
5GM
STUDY DESIGN: schematic of fish sampling
FISH
Sample Size:
200
Pathogenic Microbes:
6 organs: Skin, Gill, Liver,
Kidney, Spleen, Intestine/Stomach
Culture in BA, MacConkey Agar
Gram stain, Biochemical tests
Bacteriology:
Microbes of Public Health Importance:
at 25 C & 35 C
10 Serial dilutions, Aerobic Plate Count in Nutrient Agar at
25ºC and 35ºC
Bacteriology:
Microbes of Public Health Importance:
at 25 C& 35 C
Pathology
Gross pathology
Histopathology
6 organs: Skin, Gill, Liver,
Kidney, Spleen, Intestine/Stomach
CONT. MATERIAL AND METHODSGROSS AND HISTOPATHOLOGYGROSS PATHOLOGY: ALL THE FISH SAMPLED ASSESSED
TISSUES PROCESSED FOR HISTOLOGY ACCORDING TO LUNA (1968)
WATER SAMPLE COLLECTION POINTS
• 0M ONSHORE
• 100M OFFSHORE
• 200M OFFSHORE
LANDING SITE
• 100M BEFORE ONSHORE
• 0M ONSHORE (INLET)
• 100M OFFSHORE
• 200M OFFSHORE
RIVERS
Sample collectionWater FOR MICROBIAL INVESTIGATION
BACTERIOLOGY AND PROTOZOA INVESTIGATION
COLLECTED 5ML BELOW WATER SURFACE WITH AUTOCLAVED 250 ML BOTTLES
3 samples at landing site (onshore, 100 offshore AND 200 OFFSHORE)
4 SAMPLES AT RIVERS: 100 BEFORE ONSHORE, ONSHORE, 100 offshore AND 200 OFFSHORE)
WATER STORED AT 4̊C UNTIL PROCESSING AT A LAB
Schematics of water sampling
Water
At
4° C
Microbial pathogens
MycologySDA,
Potato Dextrose Agar
Bacteriology
10 Serial dilutions, Aerobic
Plate Count in Nutrient Agar
at 25ºC and 35ºC
Culture in BA, MacConkey
Agar
Gram stain, Biochemical
tests
Endocrine Disrupting
Chemicals
EDC extraction,
ELISA
BacteriologyB
AC
TER
IOLO
GY
BLOOD AGAR
GRAM STAIN
SUB-CULTURE
BIOCHEMICAL
TESTS (e.g. IMViC)
Anti-Microbial Resistance:
Antibiotics &
Disinfectants
MAC-CONKEY SELENITE F
NUTRIENT AGAR APC
Anti-microbial resistance
Antibiotics
• Ampicillin, Chloramphenicol, Cefuroxime, Ciprofloxacin, Erythromycin, Cetriazone, Cloxacillin, Co-trimaxazole/Trimethoprime, Nalidixic acid.
Disinfectants• Omnicide®, Lysol®, and Jik®, Brompsept, Lavik®
and Kleenol ®, and an antiseptics, Dettol ® or Savlon®.
Modified controlled disk diffusion technique (Bebora (1987) on Mueller-Hinton agar
Sample collectionWater FOR EDC INVESTIGATION
WATER COLLECTED IN 2.5 LITRE WINCHESTER BOTTLES
STORED AT 4 ̊ C UNTIL PROCESSING AT A LAB
EXTRACTION USING SPE 18 COLOMNS
ELISA EXTRACTION WITH SPECIFIC ESTROGEN KITS
CONT. M&M Endocrine Disrupting Chemicals
EDCs
Procedure: Modified pool (2007)
4 °C
WATER
COLLECTED
SPE 18
COLOMNS
EXTRACTION
ELISA KITS FOR
EI, E2, EE2, BPA
ELISA
MATERIAL AND METHODS BACTERIOLOGY
Water and fish organs (6)- transported at 4 degrees centigrade ice
Water (10 serial dilutions)
Fish organs (15 serial dilutions)–skin swabs (saline),
gills, liver, kidney, spleen, intestine (5gms)- ground with mortar and pestle, in saline
0.1ml: 0.9 ml
BACTERIOLOGYTotal BACTERIAL COUNT- cfu/gm, cfu/ml or cfu/cm2 (nutrient agar)
Blood agar, mac-conkey, selenite mac-conkey
CHARACTERISATION (gram stain, biochemical tests)
Biochemical tests:
IMViC, urease, TSI, glucose
string test (vibrio cholera),
E. coli 0157; H7 (latex agglutination test)
CONT. MATERIAL AND METHODSProtozoa:
Species of interest: Cryptosporidium, Giardia & Amoeba
Cryptosporidium: Safranin- Methylene Blue protocol
Giardia: FLOATATION method
Amoeba: STAINING WITH IODINE
Done according to who manual of parasitology (WHO, 1991)
Data collection and analysis Data will be entered into MS excel and later imported into the SAS v9.2 © 2008 (SAS institute Inc., Cary, NC, USA) for analysis.
The proportions of bacteria resistant to various disinfectants will compared between the various disinfectant dilutions
Difference in the mean total estrogenic endocrine disruptors will be analyzed by one way ANOVA and graphical presentations of the mean scores created with MS excel.
7.0 WORK PLAN
ActivityQUARTER
1
QUARTER 2 QUATER 3 QUARTER 4 QUARTER
5
QUARTER
6
QUARTER
7
QUARTER 8 QUARTER
9
QUARTER
10
QUARTER
11
Proposal
development
Field work:
Water and fish
sample collection
Lab work:
Bacteria culture,
EDC extraction
and ELISA.
Data analysis
Thesis writing
Thesis submission
BUDGETItem
1. Boat hire 18,000.00
1. Ice 5,000.00
1. Local transport 9,000.00
1. Consumables 2,000.00
1. Purchase of fish 25,000.00
1. Transport 50,000.00
1. Per Diems153,000.00
1. Gross & Histology Items 25,630.00
1. Bacteriology127, 210.00
1. EDC extraction and ELISA200,800.00
1. Miscellaneous Expenses40,000.00
Totals 528,430.00