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

Study 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

Thank You For Listening