EPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF AEROMONAS SPECIES ISOLATED FROM FISHES, POULTRY AND DIARRHOEIC PATIENTS IN ZARIA, NIGERIA
EPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF AEROMONAS SPECIES ISOLATED FROM FISHES, POULTRY AND DIARRHOEIC PATIENTS IN ZARIA, NIGERIA
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Date
2007-12
Authors
SAMUEL, SAMUEL
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for your prayers, concern and for sending me to school, thus, showing me the right way
to success. My appreciation goes to my immediate senior brother Mr. Luka Mailafia
(Debt Management Office, Abuja) for his immense concern, advice and support for my
progress. I also thank my elder brother, Dr. Obadiah Mailafia, immediate past Deputy
Governor, economic policy in the Nigeria’s apex (Central) bank, Abuja for paving way of
success for us. I am also grateful to Mr. Elie Mailafia for his concern about my progress.
My appreciation goes to my senior sisters, Mrs. Julie B. Shamah and Mrs. Sarah S. Anye
for their concern and advice. I also appreciate the concern of my younger ones, Barrister
Abel Mailafia, Barrister Nuhu Anche Mailafia and Miss Hajara Mailafia, I wish you the
best in all your endeavors.
To friends and close associates in persons of Dr. Nehemiah Yila, Dr. P.H.
Bamaiyi, Dr. Sule Adamu Garba, Dr. Asabe Dzikwi, Dr. S.J. Sambo, Dr. Obadiah Byanet
and Mrs. Ayah Ladi Addo. Thanks for your concern about my progress.
I immensely thank the management and staff of Rabboni computers, ABU press,
and Abdulrahman for sparing time and energy in typing this manuscript. I finally give
praise and honour to the most high Almighty God. He has been my guide and my
protector and by his grace, I will live.
9
ABSTRACT
Bacteriological examination of feacal samples for isolation and identification of
Aeromonas species was carried out in order to characterized them by molecular
techniques. A total of 1,160 samples obtained from 400 fishes, 540 poultry and 220
diarrhoeic patients were collected in Zaria, Nigeria between October, 2005 and January
2006.
Fishes comprising Tilapia zillii, Schilbe mystus, Synodontis filamentosus, Alestes nurse,
Marcusenius senegalensis and Clarias gariepinus were collected from 4 different
locations (A.B.U. dam, Zaria dam, Samaru market and Sabon gari market). Poultry
included layers (Brown harco) and Broilers (Anak giant) were collected from 5 areas
(Zumunta farms, Lape farms, Zaria farms, Area ‘C’ and Area ‘F’). Human samples were
collected from 2 locations (ABU Teaching Hospital and ABU sick bay).
The samples were examined for Aeromonas species using the bacteriological culture
method of isolation. Of the 1,160 samples examined from fishes, poultry and humans,
195 (16.8 %) yielded Aeromonas isolates. 84 (21 %) of the isolates were obtained from
fish, out of which 24(23.76 %) were from Tilapia zillii, 11(16.41 %) from Schilbe mystus
18(22.78 %) from Synodontis filamentosus, 14(20.89 %) from Clarias gariepinus,
17(21.25 %) from Marcusenius senegalensis and none from Alestes nurse. The results
did show that 82 (15.18 %) of the Aeromonas isolates obtained from poultry 67 (15.22
%) were from Layers and 15 (15 %) were from broilers. The least number 29 (13.8 %) of
isolates were recovered from diarrhoeic patients 5(17.14 %) of which were from Adult
males, 10(8.33 %) from adult females, 8(66.67 %) from young males and 6(33.33 %)
from young females.
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The isolates were characterized by both biochemical and molecular techniques their
prevalence rates and distributions were determined. In fish, the isolates were Aeromonas
hydrophila 47 (11.75 %), A. caviae 6 (1.50 %), A. sobria 12 (3.00 %) and A. salmonicida
19 (4.75 %). In poultry, the isolates were A. hydrophila 19 (3.52 %), A. caviae 23 (4.26
%), A. sobria 5 (0.93 %) and A. salmonicida 35 (6.48 %). In humans, the isolates were A.
hydrophila 15 (6.81 %), A. sobria 6 (2.73 %) and A. caviae 8 (3.64 %) and A.
salmonicida was not isolated from humans.
All the isolates were tested for hemolysis on 10 % sheep blood of which 72 (36.92 %)
produced ß-hemolysins. The distribution of ß-hemolysins amongst different species of
Aeromonas were A.hydrophila 31 (37.8 %), A.caviae 14 (37.83 %), A. sobria 9 (40.90
%) and A. salmonicida 18 (33.33 %).
The molecular characterization included extraction and quantification of soluble proteins
from 26 selected isolates using protein analysis. In poultry, virulent proteins were
A.hydrophila (3.58 g) A.caviae (4.00 g), A.salmonicida, (3.82 g) and A.sobria (0.00 g). In
fish, the virulent proteins were A.hydrophila (3.11 g), A sobria (4.63 g), A.caviae (2.95 g)
and A.salmonicida (2.74 g). Humans, the virulent proteins were A.hydrophila (4.07 g), A.
sobria (3.58 g) and A. caviae (3.99 g). Electropherogrammes of soluble proteins using
Sodium Dodecyl Polyacrilamide Gel Electrophoresis (SDS-PAGE) revealed that
Aeromonas species produced 6 types of toxins. The relative molecular weight ranges
between 35-73 kDa. Data obtained by analyzing Aeromonas protein profiles showed
correlation coefficients of 0.70 and 0.64 which indicated that two strains could not be
assigned to the same species.
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The genomic deoxyribonucleic acid (DNA) of the 26 Aeromonas species was extracted
using the Qiagen kit. The DNA extracts were of high purity, having optical density of
1.6-1.8. DNA quantification yielded total DNA in A. hydrophila (450 μg/ml) A. caviae
425 μg/ml, A. sobria (450 μg/ml) and A. salmonicida (375 μg/ml).
Electrophoresed DNA were further confirmed by digestion with rare-cutter restriction
endonucleases (Eco R1, Bam HI and Sal 1) which yielded restriction fragments (1.5 kb -
450 bp).
It is evident from this study that aeromoniasis is endemic affecting fishes, poultry and
humans in Zaria, Nigeria. The epidemiological significance of these findings is discussed.
Some observations on the zoonotic and public health implications of Aeromonas
infections are presented. Aeromonas species were found to have developed some virulent
factors which could be involved in pathogenicity. DNA analysis also demonstrates
common evolutionary origin of these organisms. Further studies on molecular typing of
Aeromonas is indicated.
Description
A DISSERTATION SUBMITTED TO THE POSTGRADUATE
SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF DOCTOR OF
PHILOSOPHY (PhD)
VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE
DEPARTMENT OF VETERINARY PUBLIC HEALTH AND
PREVENTIVE MEDICINE
FACULTY OF VETERINARY MEDICINE
AHMADU BELLO UNIVERSITY
ZARIA
NIGERIA.
DECEMBER, 2007
Keywords
EPIDEMIOLOGY, MOLECULAR, CHARACTERIZATION, AEROMONAS SPECIES, ISOLATED, FISHES,, POULTRY, POULTRY, ZARIA,