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Egyptian Journal of Aquatic Research (2015) 41, 93100

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National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research


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FULL LENGTH ARTICLE

Impact of using raw or fermented manure as sh


feed on microbial quality of water and sh
Nagham Elsaidy
a
b

a,*

, Fatma Abouelenien a, Ghada A.K. Kirrella

Department of Hygiene and Preventive Medicine, Faculty of Vet. Medicine, Kafrelsheikh University, 33156, Egypt
Department of Food Control, Faculty of Vet. Medicine, Kafrelsheikh University, 33156, Egypt

Received 31 October 2014; revised 19 January 2015; accepted 19 January 2015


Available online 6 March 2015

KEYWORDS
Chicken manure;
Fermented chicken manure;
Total bacterial count;
Total coliform count;
Salmonella

Abstract The microbial water and sh quality was assessed due to feeding of chicken manure
(CM) and fermented chicken manure (FCM) to sh in ponds, using Nile tilapia (Oreochromis niloticus) which were classied into 7 groups (G). Each group received different mixtures of CM or FCM
with sh ration (FR), 0:100, 25:75, 50:50 and 100:0 (%CM or FCM:% FR). The obtained results
revealed that total bacterial count (TBC) and total coliform count (TCC) were signicantly high at
P 6 0.05 in CM than both FCM and sh ration (FR). Escherichia coli and Salmonella were isolated
from CM but not from FCM or FR. Additionally, TBC and TCC were signicantly high at
P 6 0.05 at water and sh samples raised at CM ponds followed by FCM ponds in comparison with
FR. Both E. coli and Salmonella were isolated from water and sh raised in ponds receiving either
CM or FCM with higher incidence in those with CM. However all water and sh samples examined
were free from E-coli O157: H7. The obtained results, proved the inuence of CM on water and sh
quality and recommend the use of FCM as a bacteriologically safe sh pond fertilizer.
2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction
Worldwide more than 1 billion people rely on sh as an
important source of animal proteins (i.e., sh provides at
least 30% of their animal protein intakes) (Omojowo
and Omojasola, 2013). Fisheries and aquaculture in
Egypt are important components of the agricultural sector
and a signicant source of animal protein (Abo-Elela
et al., 2005; El-Naggar et al., 2008). Aquaculture is a real
* Corresponding author. Tel.: +20 1005468643, +20 473243453;
fax: +20 473231311.
E-mail address: nagham.elsaidy@yahoo.com (N. Elsaidy).
Peer review under responsibility of National Institute of Oceanography
and Fisheries.

tool in increasing sh production, which was achieved


through higher sh stocking density and the application
of articial feeding. Unfortunately, the cost of feeding is
enormous; therefore interest has been diverted to other
sources of enrichment of the water, such as using of animal manure.
Fish pond manuring is often used in sh farming for the
intensication of sh production by balancing the ratio
between carbon and other nutrients. The manure is directly
consumed by sh, and the released nutrients support the
growth of mainly photosynthetic organisms (Moav et al.,
1977; Little and Edwards, 1999). Additionally the manures
were applied to produce some necessary plant nutrients which
serve as a soil fertilizer by adding the organic matter (Sloan
et al., 2003).

http://dx.doi.org/10.1016/j.ejar.2015.01.002
1687-4285 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

94
Different kinds of manure can be utilized; cow, poultry
dung and semi-liquid pig manure are of the highest interest
(Govind et al., 1978; Wohlfarth and Schroeder, 1979).
Among manures used, chicken is preferred because of its ready
solubility and high level of phosphorus concentrations (KnudHansen et al., 1991). However, the use of manure, classied as
hazardous organic matter originating from animal feces, poses
a risk to the water environment (Mlejnkova and Sovova,
2012). Additionally it represents great public health concern;
owing to an increase in the concentrations of pathogenic
microorganisms in animal manure which is exaggerated by
the connement of farming system units with little exchange
of water (Petersen et al., 2002).
Prithwiraj et al. (2008) tested the effectiveness of introducing live zooplankton against direct manuring in ornamental
sh, through comparing four management regimes including
poultry manure (PM), live zooplankton , cow dung (CD)
and sh feed, and they found that average counts of
heterotrophic bacteria in the water of PM and CD ponds were
signicantly higher than other treatments (P < 0.05).
The microbiological analyses revealed the presence of various pathogenic microorganisms in manure in addition to the
common microora of animal intestines. Persistence of pathogens in manures and in the water environment is one of the
most important factors for infection transmission. It was found
that zoonotic pathogens can survive in such environments up to
4 months, depending on the type of manure, temperature, pH,
oxygen level, ammonia concentration and the presence of competing organisms (Jones, 1976; Guan and Holley, 2003).
Under the use of thermophilic fermentation of CM most of
pathogens are destroyed. The thermophilic anaerobic process is
effective against pathogenic bacteria (such as fecal coliform,
salmonella and enterococcus) (Watanabe et al., 1997).
Salmonella and Mycobacterium paratuberculosis were inactivated within 24 h under thermophilic conditions, while weeks or
even months will be needed under mesophilic conditions
(Sahlstrom, 2003).
Information about the effect of manuring on water quality
and fecal contamination is scarce. Additionally the usual practice of sh pond owners to keep CM in tightly sealed plastic
bags and leave it for some time in the sun, creates a condition
resembling to some extent the anaerobic thermophilic fermentation process. So the aim of this study is to clarify the impact
of using raw CM in sh farm environments on the occurrence
of water pollution indicator bacteria, dening related potential
health risks represented by pathogenic bacterial contamination
especially the detection of Salmonella and E. coli O157.
Moreover, the effect of anaerobic treatment of CM under
thermophilic conditions on microbial contamination of sh
pond water has to our knowledge not been investigated
previously.
Material and methods
This study was carried out for 60 days (from 1st September till
1st November, 2012) at the Laboratory of the Department of
Hygiene and Preventive Medicine, Faculty of Vet. Medicine,
Kafr El-Shiekh University, Egypt. Nile tilapia (Oreochromis
niloticus), were used for this study.
Fingerlings with a mean average weight of 10 0.58 g and
7 0.68 cm length were obtained from the local sh farm (ElReyad, Kafr El-Shiekh, Egypt). Fish were homogeneous in size

N. Elsaidy et al.
and body weight and apparently healthy. The sh were acclimatized for 7 days before the experiment. They were fed on
the same diet used in this study. During this adaptation period,
the dead and weak sh were eliminated daily.
Chicken manure (CM) and fermented chicken manure (FCM)
CM was obtained from the Kafr El-Shiekh University chicken
farm (cage layer system) and was collected from deposits
directly under chicken cages. Fermented CM (FCM) was prepared by placing CM in a set of 125 ml capacity anaerobic
serum bottles. The headspaces of the bottles were purged with
N2 gas, and the vials were sealed with rubber stoppers and
crimped aluminum caps. These bottles were incubated
anaerobically at 55 C. Biogas produced was monitored every
day. When gas production stopped, vials were opened; FCM
produced was collected and kept under 20 C until use
(Abouelenien et al., 2009, 2010).
Experimental design
Feeding regime of 3% of Body.wt per day was employed
throughout the trial. The amount of feed was calculated and
readjusted weekly. The sh aquaria under experiment were
divided into 7 groups (G), stocked into 14 aquaria (dimensions,
40 50 100 cm) at a stocking rate of 14 sh per aquaria (60 L
water/aquaria,). The sh aquaria were supplied with chlorine
free tap water with continuous aeration, using an electric aquarium air pump (Hali BAO-Cx 8200) for 60 days. Two sets of
manure feeding were used, the rst set received CM as a feed
with different ratios (ranged from 0% to 100%), the second
set received FCM, with different ratios (0100%), and the control aquaria which received commercial sh ration (FR). 2 replicates for each treatment were made, with water changes on a
daily basis. A detailed description of the groups is illustrated
in Table 1.
Sampling
CM, FCM and FR were sampled with weights of 100 g, each
sample was transferred into a plastic bag and analyzed to enumerate total bacterial and fecal counts, as well as to determine
the presence of Escherichia coli and Salmonella spp. (APHA,
1992). Water sampling was done once every 2 weeks, with a
total number of 4 water samples which was done at a xed
time (Jha et al., 2004). Water samples were collected by inverting a 250 ml sterilized glass bottle 30 cm below the pond water
surface. All samples were transferred to the lab in an icebox.
Analysis was initiated within 2 h of sample collection. Five sh
were sampled at the same time with water sampling by harvesting with a net and put in a sterile polyethylene bag. 10 g of the
muscle portion of sh along with the skin was homogenized
for 1 min with 90 ml of peptone water in a homogenizer
(PolytronPT-MR 2100).
Bacteriological analysis
Enumeration of total bacterial counts (TBCs)
The total bacterial count of water samples was carried out
according to Cruickshank et al. (1972) and Ayandirana et al.
(2014).

Impact of fermented chicken manure on microbial quality of water and sh


Table 1

95

Description of experiment groups, sh ration (FR), chicken manure (CM) and fermented chicken manure (FCM).

Group number (G)

G1
G2
G3
G4
G5
G6
G7

Type of treatment

Control
25% CM
50% CM
100% CM
25% FCM
50% FCM
100% FCM

No. of Aquaria

2
2
2
2
2
2
2

The total bacterial count of sh samples was carried out


according to APHA (1992).

Feeding mixture
Fish ration (FR) (%)

CM (%)

FCM (%)

100
75
50
0
75
50
0

0
25
50
100
0
0
0

0
0
0
0
25
50
100

prepared nutrient agar plates to obtain pure isolate cultures


of bacteria. Pure isolates of Salmonella sp. were subjected to
the IMViC test, Motility test, and the Urease activity test.

Estimation of total coliform count (TCC)


Detection of Salmonella in sh samples
Total coliform of pond water and sh samples was enumerated
by a three tube MPN procedure (APHA, 2005). Aliquots of
serially diluted samples were inoculated into MacConkey
broth (Oxoid, UK) and incubated at 37 C for 2448 h.
Positive tubes were (1) subjected to an MPN procedure in brilliant green lactose bile broth (Oxoid, UK) and incubated at
37 C for 2448 h. All tubes showed the production of acid
and gas within each group which were recorded. The relative
MPN index of coliform organisms per 100 ml of sample was
obtained according to APHA (1998). and subjected to indole
broth at 44.5 C. A loopful of bacterial culture from indole
positive tubes was streaked on Eosin Methylene Blue agar
(LABM, UK) and characteristic E-coli colonies were isolated
and conrmed by performing IMViC tests where, I is
Indole production from Tryptone broth, M and V are
Methyle red and the Voges poskauer test from Buffered
Glucose broth and C is the Citrate utilization test from
Simmons Citrate agar (APHA, 2001).
Isolation and identication of E. coli O157: H7
According to Okrend et al. (1990), (a) Selective enrichment:
25 ml of water samples and 25 g of the examined sh samples
were weighed aseptically into a sterile container and thoroughly homogenized with 225 ml of MTSB broth (Modied
Tryptone-Soya broth), incubated at 42 C for 24 h. (b)
Selective plating, a loopful from selective enrichment broth
was streaked into Harlequin SMAC-BCIG media (Sorbitol
McConkey Agar with BCIG) and Incubated at 37 C for
24 h in which suspect colonies appear translucent; (c)
Biochemical conrmation (FDA, 1998), the suspected colonies
were puried and identied using the IMVIC pattern. (d)
Serological identication, the suspected E. coli O157: H7 were
serologically conrmed using IGM antibodies to O157: H7
(E. coli O157: H7 test kit, Oxoid) according to the
manufacturer.
Detection of Salmonella
Detection of Salmonella in water samples was checked by
enrichment in selenite broth and isolation and conrmation
on xylose lysine deoxycholate (XLD) agar plates (Polo et al.,
1998). Typical colonies of the Salmonella sp., from selective
agar were streaked aseptically several times on to freshly

According to ISO (2002), (a) Pre-enrichment, 25 g of the examined samples was weighed aseptically into a sterile container
and homogenized with 25 ml of sterile buffered peptone,
water, incubated at 37 C for 24 h; (b) Selective enrichment,
1 ml of incubated pre-enrichment homogenate was transferred
to 10 ml Muller-Kauffmann tetrathionate novobiocin broth,
incubated at 37 C for 24 h; (c) Selective plating, a loopful from
the selective enrichment broth was streaked into the XLD
media, incubated at 37 C for 24 h. Suspected colonies appear
green in color; (d) Biochemical conrmation (FDA, 1998), the
suspected colonies were puried and identied using the
IMVIC pattern.
Statistical analysis
Data were tested for distribution normality and homogeneity
of variance. Data were reported as mean SEM and analyzed
by a one-way ANOVA using Minitab15 (Minitab Inc., State
College, PA, USA). The signicance of difference among the
different treatments was evaluated by the Tukey test. The signicance level was set at P 6 0.05.
Results and discussion
The microbial quality of pond water and sh samples in this
study was described by indicator microorganism detection,
including total bacterial count (TBC), total coliform count
(TCC) and E. coli (EC). Also, the potential health risk represented by pathogenic bacterial contamination was proved by
detection of Salmonella and E. coli O157.
Bacterial load in FR, CM and FCM
Results in Table 2 reveal that, total bacterial count (log10 cfu/
g) showed no signicant difference at (P 6 0.05) between raw
chicken manure (CM) and fermented chicken manure
(FCM), while they were signicantly different at (P 6 0.05)
from Commercial sh ration (FR), where the mean values of
total bacterial count (TBC) for FR, CM and FCM were
4.07, 8.59 and 6.50 log10 cfu/g respectively. On other hand,
total coliform count (TCC) showed a signicant difference at
(P 6 0.05) between FR, CM and FCM, where the mean values
were 0.98, 3.39 and 1.4 log10 cfu/g respectively.

96

N. Elsaidy et al.

Table 2 Microbiological quality of sh ration (FR), raw chicken manure (CM) and fermented chicken manure (FCM) used in the
experiment (mean SE).
Bacteriological analysis

FR

CM

FCM

P-value

Total bacterial count (log10 CFU/g)


Total coliforms (log10 CFU/g)
P/A of (E-coli/100 g)
P/A of Salmonella/100 g)

4.07 0.24a
0.98 0.11a

8.59 1.2b
3.39 0.02c
+
+

6.50 0.76b
1.4 0.09b

0.002
0.001

Mean values in each row having a common superscript are non-signicantly different at p 6 0.05.
P/A = presence or absence.
+ = Presence of Salmonella and E-coli.
= Absence of Salmonella and E-coli.

These results explained that TBC and TCC were much


higher at CM, followed by FCM in comparison with FR,
where these results agreed with those given by Omojowo and
Omojasola (2013). The manure can provide a favorable environment for pathogen survival and even re-growth due to
the availability of nutrients as well as protection from UV
radiation, desiccation, and temperature extremes (Rogers
and Haines, 2005). As properly handled and treated manure
is an effective and safe fertilizer, untreated or improperly treated manure may become a source of pathogens that may contaminate soil, fresh products, surface and ground water and
drinking water supplies (Vanotti et al., 2007). From these
results it was clear that CM subjected to fermentation under
thermophilic condition (55 C) resulted in a decrease in TBC
and TCC.
Many pathogenic bacteria are present in animal manures.
The bacterial pathogens most important with regard to human
health include, for example, Salmonella sp., E. coli O157 H7
(Venglovsky et al., 2006). Bacterial pathogens such as
Salmonella and E. coli O157:H7 can survive for several months
in manure when environmental conditions are favorable (low
temperatures, good moisture level) (Rogers and Haines,
2005). Table 2 explains that E-coli and Salmonella were isolated only from raw CM and not isolated from either FR or
FCM; similar results are obtained by Machado et al. (2006),
which come with the fact that exposure of CM to thermophilic
anaerobic fermentation kills most bacterial pathogens by exposure to temperatures of 5566 C for a few hours or less
(Epstein, 1997; De Leon and Jenkins, 2002), temperature in
particular is a critical factor in pathogen survival, with cooler
temperatures generally enabling longer survival times.
Bacterial load of water samples
In Table 3 results show, there was no signicant difference at
P 6 0.05 in the total bacterial count (log10 cfu/ml) between different manure treated ponds either CM or FCM, however the
highest (TBC) was recorded in ponds that received CM than
FCM but both of them were signicantly different at P 6 0.05
from control ponds (FR). Results revealed that TBC was least
at the ponds that received 25% FCM (3.90), followed by ponds
that received 50% FCM (4.03) log10 cfu/ml. On other hand, the
results were 3.92, 3.98 and 4.33 log10 cfu/ml at ponds that
received 25%, 50% and 100% CM, respectively.
These results were nearly similar to those given by
Prithwiraj et al. (2008), Mlejnkova and Sovova (2012) and
Omojowo and Omojasola (2013). The increase of TBC in
ponds in relation to an increase in the ratio of CM used may

be attributed to the increased input of easy biodegradable


organic matter and bacterial inoculum with an increase in
the ratio of CM. However, the results are lower than the recommended total bacterial count in source water of sh according to WHO (1989).
In addition, Table 3 shows that TCC was not signicantly
different at (P 6 0.05) at all manure treated ponds and control
ponds, however results showed the highest count was recorded
at ponds that received CM (0.88, 1.02 and 1.00 log10 cfu/
100 ml at 25%, 50% and 100% CM respectively), than ponds
that received FCM, where results were 0.94 and 0.90 log10 cfu/
100 ml for 25% and 50% FCM, respectively. Similar results
were given by Omojowo and Omojasola (2013). However the
results are comparably lower than the natural manure fertilized sh cultured ponds stocked with carp, tilapia etc.,
(Quines, 1988; Pilarski et al., 2004) and lower than the recommended coliform count in source water of sh according to
WHO (1989), where they may be attributed to the low number
of reared sh and frequent changes of water as compared with
the natural condition of rearing of such cultured sh. G 7
(FCM 100%) results are not included as 100% mortality rate
occurred, which was attributed to ammonia toxicity (level of
ammonia 2869 ppm results under publication) detected in the
two aquaria of this group.
Bacterial load at sh samples
Fish are conditioned by their environment and hence it is obvious that if the growing and harvesting environment of sh is
polluted chemically or microbiologically, the sh are also polluted (Boyd, 1984). Total plate count is a good indicator of
the sensory quality or expected shelf life of the product
(Olafsdottir et al., 2006; Koutsoumanis and Nychas, 2000)
and to be considered safe, TBC of sh should never exceed 7
log cfu/g wet weight (ICMSF, 1986).
The results of sh samples presented in Table 4 reveal the
presence of a signicant difference at (P 6 0.05) among treated
groups FR, CM and FCM where TBC ranged from 1.02, 1.27
and 1.64 log10 cfu/g for sh raised in ponds fertilized by 25%,
50% and 100% CM, respectively which recorded the highest
count, while it was 1.08 and 1.35 log10 cfu/g for sh raised in
ponds fertilized by 25% and 50% FCM, respectively. Also
TCC for sh raised in fertilized ponds with 25%, 50% and
100% CM were 0.53, 0.87 and 0.79 log10 cfu/g, respectively
and were 0.75 and 0.76 log10 cfu/g for 25% and 50% FCM,
respectively. These results are in agreement with Chowdhury
et al. (1989) and Al Harbi (2003), who observed that bacterial
load in sh samples was correlated with the bacterial levels in

Impact of fermented chicken manure on microbial quality of water and sh


Table 3

97

Microbiological quality of water samples of different treatment groups used in the experiment (mean SE).

Bacteriological analysis

G1

G2

G3

G4

G5

G6

P-value

TBC (log10 cfu/ml)


TCC (log10 cfu/100 ml)
E-coli O157

2.73 0.48a
0.49 0.2
ND

3.92 0.35b
0.88 0.30
ND

3.98 0.15b
1.02 0.15
ND

4.33 0.16b
1.00 0.47
ND

3.90 0.28b
0.94 0.54
ND

4.03 0.39b
0.90 0.39
ND

0.001
0.182

Mean values in each row having a common superscript are non-signicantly different at P 6 0.05.
G1, G2, G3, G4, G5 and G6 as depicted in Material and methods.
ND = Not detected.

Table 4

Microbiological quality of sh samples of different treatment groups used in the experiment (mean SE).

Bacteriological analysis

G1

G2
a

TBC (log10 cfu/g)


TCC (log10 cfu/g)
E-coli O157

G3
a

0.79 0.17
0.30 0.01a
ND

G4
ab

1.02 0.03
0.53 0.04ab
ND

G5
b

1.27 0.18
0.87 0.08b
ND

G6
a

1.64 0.28
0.79 0.29ab
ND

P-value
ab

1.08 0.0
0.75 0.2ab
ND

1.35 0.39
0.76 0.19ab
ND

0.008
0.017

Mean values in each row having a common superscript are non-signicantly different at P 6 0.05.
G1, G2, G3, G4, G5 and G6 as depicted in Materials and methods.
ND = Not detected.

the aquatic environment. These results agreed with Dritan and


Bejo (2013) who reported that the use of untreated livestock
manure releases high concentrations of pathogenic microorganisms into the ponds constituting a high risk to sh and sh
farmers. However these results are lower than those given by
Omojowo and Omojasola (2013) and the recommended level
is described by ICMSF (1986). Signicantly, the sh from
the FCM fertilized ponds had a better microbial quality which
explains the advantage of using fermented chicken manure
over raw chicken manure as at thermophilic anaerobic fermentation most bacterial pathogens are killed by exposure to temperatures of 5566 C for a few hours or less (Epstein, 1997;
De Leon and Jenkins, 2002).

Table 5, reveals that the incidence of the isolated E-coli and


Salmonella sp. in water samples was increased with an increase
in the ratio of CM used which was 0%, 33% and 50% of isolates for E-coli at 25%, 50% and 100% CM respectively that
agreed with Dritan and Bejo (2013) who reported that among
144 isolates from the Poultry manure identied, E-coli represents about 62 (43%), which explains the increasing incidence
of isolated E-coli from pond water with an increase the input
ratio of CM. Also the result of Salmonella was 50%, 60%
and 100% of isolates at 25%, 50% and 100% CM, respectively,
however there was a lower incidence in groups that received
FCM in comparison with those that received CM which
showed 40% and 11% for E-coli at 25% and 50% FCM and
33% for both 25% and 50% FCM for the Salmonella sp.
Similar results were obtained by Ogbondeminu and Okaeme
(1989) who reported that 50% of the microorganisms recovered
from both sh and water of earthen ponds fertilized with animal fecal waste were members of the family
Enterobacteriaceae. On the other hand the control ponds
(FR) revealed the presence of one isolate for E-coli which
may be attributed to the E-coli reported as predominant enteric
bacteria from intestines of fresh water shes (Carp, tilapia and
catsh) and pond water (Niemi and Taipalinen, 1982;
Ogbondeminu, 1993). However, the presence of typical E-coli
in water was considered as the most reliable index of excretal
pollution, with consequent liability of contamination with
other forms of pathogenic organisms (Cruickshank, 1962;
Sabae and Rabeh, 2007).

Incidence of E-coli and Salmonella in water and sh samples


Human infections commonly caused by pathogens are transmitted from sh or the aquatic environment. Facultative bacterial pathogens for both sh and man may be isolated from
sh without apparent symptoms of disease (Acha and
Szyfres, 2003).
Aquatic environments are the major reservoirs of
Salmonella. Therefore, shery products have been recognized
as a major carrier of food-borne pathogens (Kamat et al.,
2005; Upadhyay et al., 2010). Approximately 95% of human
salmonellosis cases are associated with the consumption of
contaminated products such as meat, poultry, eggs, milk and
seafood (Ibrahim et al., 2014).

Table 5
M.O

Incidence of E-coli and Salmonella in water samples.


Groups
G1

Salmonella
E-coli

G2

G3

G4

G5

G6

Total

No.

+ve

No.

+ve

No.

+ve

No.

+ve

No.

+ve

No.

+ve

No.

+ve

2
3

0
1

0
33

4
2

2
0

50
0

5
3

3
1

60
33

2
6

2
3

100
50

3
5

1
2

33
40

3
3

1
1

33
33

19
22

9
8

47
36

G1, G2, G3, G4, G5 and G6 as depicted in Materials and methods.

98
Table 6
M.O

N. Elsaidy et al.
Incidence of E-coli and Salmonella in sh samples.
Groups
G1

Salmonella
E-coli

G2

G3

G4

G5

G6

Total

No.

+ve

No.

+ve

No.

+ve

No.

+ve

No.

+ve

No.

+ve

No.

+ve

1
2

0
0

3
1

0
0

2
2

1
0

50

2
5

1
2

50
40

1
3

0
1

33

1
2

0
0

10
15

2
3

20
20

G1, G2, G3, G4, G5 and G6 as depicted in Materials and methods.

Results presented in Table 6, indicate that both E-coli and


Salmonella were isolated from sh raised in both ponds fertilized with CM and FCM with a higher incidence at ponds fertilized by CM especially for E-coli. These results agreed with
those obtained by Nair and Nair (1988), Karunasagar et al.
(1992), Nedoluha and Westhoff (1997), Muratori et al.
(2000), who reported isolation of different members of
Enterobacteriaceae as potential sh and human pathogens
from natural manured carps, stripped bass, tilapia, eel and
its earthen culture environment. Their occurrence was much
higher in integrated sh culture systems (Ogbondeminu,
1993). High levels of fecal coliform, E-coli and Salmonella in
pond water lead to breakage of the immunological barrier of
sh and invasion of sh muscles by pathogens (Guzman
et al., 2004).
Isolation of E-coli O157:H7 from water and sh samples
The quality of fresh sh is strongly determined for bacterial
microbiota (Gram, 1992). In this context, the use of E. coli
as a sanitary indicator for sh samples has been rst reported
in the 1930s (Grifths and Fuller, 1936). E. coli O157:H7 could
be present in water contaminated by sanitary sewer overow
or runoff from farm elds. Additionally there are reports of
the occurrence of pathogenic strains of E-coli from market
sea food from different parts of the world (Samadpour et al.,
1994; Teophilo et al., 2002). Fortunately it is clear from the
ndings of the present study that all examined water and sh
samples were free from the emerging pathogen E-coli
O157:H7,which may be similar to those results obtained by
Surendrarai et al. (2009) and Mlejnkova and Sovova (2012).
Conclusion
Bacteriological analysis revealed that TBC and TCC, was
much higher in CM than FCM and FR. E-coli and
Salmonella were isolated only from CM and not isolated from
FCM or FR of sh. Bacteriological analysis of water and sh
samples revealed that the bacterial load of water and sh samples of sh ponds that received fermented chicken manure was
much lower than the bacterial load in ponds that received raw
chicken manure.
Also E-coli and Salmonella were isolated with high incidence in ponds that received CM than ponds that received
FCM, although all samples of water and sh studied were free
from the emerging pathogen E-coli O157:H7.
As excessive use of raw CM may present a risk related to
increased supply of nutrients, decay of organic matter and the
presence of enteric pathogens. FCM resulted from thermophilic

anaerobic fermentation of CM at 55 C which was used primarily to convert waste to energy, and was more bacteriologically safe as a sh pond fertilizer than using raw CM. Our results
draw attention to the need of pretreatment of CM before use
as a sh pond fertilizer in order to avoid infection by such pathogens. Also sh raised in sh ponds fertilized by animal manure
should be properly cooked before human consumption.
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