Download pathogenic microbes and endocrine disrupting chemicals in water

PATHOGENIC MICROBES AND
ENDOCRINE DISRUPTING CHEMICALS IN
WATER AND OREOCHROMIS NILOTICUS
IN 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)
INTRODUCTION
The 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 REVIEW
Increased 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
JUSTIFICATION
Fish 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
Study site
Sample Sizes
Formula 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 COLLECTION
FISH SAMPLES FOR MICROBIAL
MICROBIAL INVESTIGATION
SKIN
SWAB
1 INCH SQ
STOMACH
GILL
SWAB
5GM
FISH
SPLEEN
KIDNEY
5GM
5GM
LIVER
5GM
stored in saline and transport media at 4 ̊C until processing at a lab
STUDY DESIGN: schematic of fish sampling
Pathogenic Microbes:
10 Serial dilutions, Aerobic
Plate Count in Nutrient Agar at
25ºC and 35ºC
Bacteriology:
Microbes of Public Health
Importance:
at 25 C& 35 C
6 organs: Skin, Gill, Liver,
Kidney, Spleen,
Intestine/Stomach
Culture in BA, MacConkey
Agar
FISH
Gram stain, Biochemical tests
Sample Size:
Bacteriology:
Microbes of Public Health
Importance:
at 25 C & 35 C
200
Gross pathology
Pathology
6 organs: Skin, Gill, Liver,
Histopathology
Kidney, Spleen,
Intestine/Stomach
CONT. MATERIAL AND METHODS
GROSS AND HISTOPATHOLOGY
GROSS PATHOLOGY: ALL THE FISH SAMPLED ASSESSED
TISSUES PROCESSED FOR HISTOLOGY ACCORDING TO LUNA (1968)
WATER SAMPLE COLLECTION POINTS
LANDING
SITE
RIVERS
• 0M ONSHORE
• 100M OFFSHORE
• 200M OFFSHORE
• 100M BEFORE ONSHORE
• 0M ONSHORE (INLET)
• 100M OFFSHORE
• 200M OFFSHORE
Sample collection
Water 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
Mycology
SDA,
Potato Dextrose Agar
10 Serial dilutions, Aerobic
Microbial pathogens
Plate Count in Nutrient Agar
at 25ºC and 35ºC
Water
Bacteriology
At
4° C
Culture in BA, MacConkey
Endocrine Disrupting
EDC extraction,
Chemicals
ELISA
Agar
Gram stain, Biochemical
tests
BACTERIOLOGY
Bacteriology
GRAM STAIN
BIOCHEMICAL
TESTS (e.g. IMViC)
BLOOD AGAR
SUB-CULTURE
Anti-Microbial Resistance:
Antibiotics &
Disinfectants
MAC-CONKEY
SELENITE F
NUTRIENT AGAR
APC
Anti-microbial resistance
Modified controlled disk diffusion technique (Bebora (1987) on Mueller-Hinton
agar
Antibiotics
Disinfectants
• Ampicillin, Chloramphenicol, Cefuroxime,
Ciprofloxacin, Erythromycin, Cetriazone,
Cloxacillin, Co-trimaxazole/Trimethoprime,
Nalidixic acid.
• Omnicide®, Lysol®, and Jik®, Brompsept, Lavik®
and Kleenol ®, and an antiseptics, Dettol ® or
Savlon®.
Sample collection
Water 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)
WATER
COLLECTED
4 °C
EXTRACTION
SPE 18
ELISA
ELISA KITS FOR
COLOMNS
EI, E2, EE2, BPA
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
BACTERIOLOGY
Total 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 METHODS
Protozoa:
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
Activity
QUARTER
1
Proposal
development
Field work:
Water and fish
sample collection
Lab work:
Bacteria culture,
EDC extraction
and ELISA.
Data analysis
Thesis writing
Thesis submission
QUARTER 2 QUATER 3
QUARTER 4 QUARTER
5
QUARTER QUARTER QUARTER 8 QUARTER
6
7
9
QUARTER QUARTER
10
11
BUDGET
Item
1.
Boat hire
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 Diems
1.
Gross & Histology Items
1.
Bacteriology
1.
EDC extraction and ELISA
1.
Miscellaneous Expenses
Totals
18,000.00
153,000.00
25,630.00
127, 210.00
200,800.00
40,000.00
528,430.00
Thank You For Listening