Fungi Load and prevalence of Aspergillus species in Meat Markets and Abattoirs in Ibadan, Oyo State

Abstract

Meat contamination in abattoirs and meat markets has been associated with several factors. Of major concern is contamination due to pathogenic microbes present in food processing environments. This study investigated fungal contamination in meat markets and abattoir environments. Four meat markets (Olunloyo, Oja Oba, Olorunsogo, and Olodo) and three abattoirs (Olorunsogo, Akinyele, and University of Ibadan) in Ibadan, Oyo State, Nigeria, were assessed for prevalence of Aspergillus species. Fungi counts were determined by Pour-plate method at 10-5 dilution on Sucrose-Potato Dextrose Agar, supplemented with streptomycin, and incubated at 26ºC for 5 days. Culture and enumeration of Aspergillus flavus and Aspergillus niger from table scrapings (n = 260) and swabs (n=100) of abattoir environment were done using standard microbiological methods. The mean value for the total fungal count (TFC) was 4.46 ± 0.24 log CFUml-1. There were significant differences in the total fungal counts among locations, with Olunloyo market having the highest fungal load (4.73 ± 0.64 log CFUml-1), and the lowest total fungal count was at the University of Ibadan abattoir (4.14 ± 1.29 log CFUml-1). A. niger (30.18 %) was the most frequently isolated fungi, while the least was A. fumigatus (1.0 %). Prevalence of A. flavus was 14.79 %. Other fungi species were A. tamarii, A. terreus, Fusarium compacticum, F. oxysporum, F. proliferum, Penicillium chrysogenum, and P. oxalicum. The study revealed a compromise in food safety in meat producing areas of Ibadan and hence, a need to enhance hygienic standards to improve food safety in these locations.

Full text

Ogundijo and
Meat contamination in abattoirs and meat markets has been associated with several factors.
major concern is contamination due to pathogenic microbes present in food
environments. This study investigated fungal contamination in meat markets and
environments. Four meat markets (Olunloyo, Oja Oba, Olorunsogo, and Olodo) and
abattoirs (Olorunsogo, Akinyele, and University of Ibadan) in Ibadan, Oyo State, Nigeria,
assessed for prevalence of Aspergillus species. Fungi counts were determined by
method at 10-5 dilution on Sucrose-Potato Dextrose Agar, supplemented with streptomycin,
incubated at 26ºC for 5 days. Culture and enumeration of Aspergillusflavus and
from table scrapings (n = 260) and swabs (n=100) of abattoir environment were done
standard microbiological methods. The mean value for the total fungal count (TFC) was 4.46 ±
0.24 log CFUml-1. There were significant differences in the total fungal counts among
with Olunloyo market having the highest fungal load (4.73 ± 0.64 log CFUml-1), and the
total fungal count was at the University of Ibadan abattoir (4.14 ± 1.29 log CFUml-1). A.
(30.18 %) was the most frequently isolated fungi, while the least was A. fumigatus (1.0
Prevalence of A. flavus was 14.79 %. Other fungi species wereA. tamarii, A. terreus,
compacticum,F. oxysporum, F. proliferum, Penicillium chrysogenum, and P. oxalicum.
study revealed a compromise in food safety in meat producing areas of Ibadan and hence, a
to enhance hygienic standards to improve food safety in these
Fungi Load and prevalence of Aspergillus species in Meat Markets
and Abattoirs in Ibadan, Oyo State
Ogundijo, O. A. and Adetunji, V. O.*
Department of Veterinary Public Health and Preventive Medicine,
Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria
*Correspondence:vadetunji@gmail.com
Key words: Aspergillus spp., Contamination, Abattoir, Food safety, Ibadan.
Introduction
Meat contamination in abattoirs and meat
stalls could result from contaminated water,
unhygienic practices like poor handling, use
of contaminated tables to display meat meant
for sale and the use of contaminated knives in
cutting operations. Contamination of meat and
meat products occurs when raw meat is
exposed or makes contact with pathogenic
microbes which are ubiquitous in nature
(Edema et al., 2005). The Food and
Agricultural Organization (FAO) and World
Health Organization (WHO) state that illness
due to contaminated food is perhaps the most
widespread health problem in the
contemporary world and an important cause of
reduced economic productivity (Edema et al.,
2005).
Serious consequences relating to
public safety can arise from lack of hygiene
and sanitation in abattoirs and meat stalls
(Fasanmi et al., 2010) which can be of
adverse effect on public health. Aspergillus
species are ubiquitous,saprophytic fungi
that play a significant role in global
carbon and nitrogen recycling (Pitt, 1994;
Haines, 1995).They are commonly found in
air, water, soil, plant debris, rotten
vegetation, manure, sawdust litter, bagasse
litter, and animal feed, on animals and in
indoor air environments (Pattron, 2006;
Bennett et al., 2010). These fungi species
have been found to be pathogenic in

Ibadan Journal of Agricultural Research Vol. 14(1), 2018
humans, being opportunistic in nature. A.
fumigatus, A. flavus, A. terreus, A. niger,
and A. nidulans have all been implicated in
human and animal infections (Denning,
1998; Morgan et al., 2005). A. flavus has
been found to cause upper respiratory tract
infections faster than any other Aspergillus
species (Kennedy et al., 1997; Panda et al.,
1998). They also cause a broad spectrum of
diseases, ranging from hypersensitive reactions
to invasive infections associated with angio-
invasion (Anand and Tiwary, 2010). Studies to
specifically assess the environmental impact of
this organism are limited when compared to
the interest of researchers in other
microorganisms. This study aimed at
determining the Fungi counts and the
prevalence of Aspergillus spp. on meat tables
and abattoirs in Ibadan, Oyo state.
Materials and Methods
Study location and sample size
This study was carried out in six locations
of five different Local Government areas
(LGA) in Ibadan, located at 7° 23' N, 3° 55'
E, with an average temperature of 23.94
o
C
and relative humidity of 74.55 %., Oyo
State, Nigeria. The selected study areas
were; Olunloyo market (Ona Ara LGA),
Oja Oba market (Ibadan South-West LGA),
Olorunsogo market (Ona Ara LGA), Olodo
market (Lagelu LGA), Akinyele slaughter
house (Akinyele LGA) and University of
Ibadan abattoir (Ibadan North LGA). Total
number of samples collected was 360
samples consisting of meat table scrapings
(n = 260) and swabs from slaughter slabs
and walls of abattoirs (n = 100).The studied
areas and samples were selected using the
random sampling technique.
Sample collection and processing
Samples of table scrapings were collected
from Olunloyo (n = 70), Oja Oba (n = 60),
Olorunsogo (n = 80), and Olodo (n = 50),
while swabs from slaughter slabs and walls
were taken from Olorunsogo (n = 20),
Akinyele (n = 40), and University of Ibadan
(n = 40). Samples of table scrapings were
collected using sterile universal bottles and
swabs were taken with sterile swab sticks.
Collected samples were placed in 9 mls of
peptone water, placed in ice packs and
transported to the laboratory for processing
within 12 hours of collection. The samples
were analysed at the Microbiology laboratory
of the Institute for Agricultural and Research
Training, Moor, Apata, Ibadan.
A ten-fold serial dilution to a factor of
five (10-5) of 1 ml of the collected samples
were prepared using 9 mls of physiological
saline solution in test tubes. Using the Pour-
plate method, 1 ml of the serially diluted
samples were cultured in 10 mls of
Sucrose-Potato Dextrose Agar
supplemented with streptomycin to prevent
other microbial growth, and incubated at 26
°C for five days. Fungal counts for each of
the plates were done using a colony counter
and pure colonies were sub-cultured in
Sabouraud dextrose agar for identification.
Fungal isolation and identification
The identification of fungal specieswas
based on gross colony morphology, colour and
on microscopic features.Distinct colonies were
stained on glass slides using Lactophenol
cotton blue for proper examination with the aid
of a photomicroscope as described by Singh et
al. (1991). This was carried out in the
Veterinary Pathology Laboratory, University
of Ibadan, Ibadan.
Statistical analysis
One-way analysis of variance (ANOVA)
was performed on the data and standard
deviations for the locations and organisms
were calculated. Significantly different means
were separated using Duncan Multiple Range
Test (p<0.05%) (Obi, 1990).
Results
The identification of all cultured organisms
was based on their macroscopic and
microscopic features on Sabouraud
dextrose agar (Plates 1 and 2) and

Ogundijo and Adetunji
photomicroscope (Plate 3 and 4). A. niger (Plate 2). At ×400 magnification, the
was identified by its blackish-brown colour,
which was cream-yellow to yellow on the conidial head of A. niger was globose, with
globose to elliptical, rough, and dark brown
reverse side of the plate on Sabouraud to black conidia (Plate 3a). For A. flavus,
dextrose agar (Plate 2), while A. flavus was the conidial head was radiating, which
identified with its characteristic yellowish- became loosely columnar with time (Plates
green to green colour which was cream- 3b and 4) (Singh et al., 1991).
yellow on the reverse side of the plate
Plate 1. Mixed cultures showing Penicillium oxalicum (left) and Fusarium oxysporum
(right)on Sucrose-Potato dextrose agar (Aspect Ratio: 4: 3).
A B
Plate 2. Pure culture
plates showing A. niger (A)and
A. flavus (B) on Sabouraud
Dextrose agar (Aspect Ratio: 4: 3).
A
Plate 3. The globose conidial head of A. niger (A) (×40 magnification) and radiating
conidial head of A. flavus (B) (×400 magnification), using Lactophenol cotton
blue staining technique.
B

Ibadan Journal of Agricultural Research Vol. 14(1), 2018
Plate 4. The radiating conidial heads of A. flavus, with sub-globose, rough and yellowish-
green conidia (×400 magnification).
A total of ten (10) fungal organisms were (18%) and the least was found in UI
isolated and identified. The isolates include; abattoir (13.6 %) (Table 2). A. niger, A.
Aspergillus flavus, A. fumigatus, A. niger, A.
tamarii,A. terreus, Fusarium compacticum, F. flavus and F. oxisporum where the highest
contaminating fungi. The mean total fungal
oxysporum,F. proliferum, Penicillium count (TFC) was 4.46 ± 0.24 (Table 3). The
chrysogenum,and P. oxalicum (Table 1). The
most frequently encountered Fungi was A. total fungal load for Olunloyo market (4.73
± 0.64) was found to be the highest, and the
niger (30 %), followed by F. oxisporumwhile lowest total fungal count was at the
the least frequently encountered in this study
was A. fumigatus (1 %) (Table 1).
Olodo market (18.9 %) had the highest
fungal contamination,followed by Oja oba
University of Ibadan (UI) abattoir (4.14 +
1.29).
Table 1. Percentage Occurrence of different fungal species
Fungus
Frequency
Percentage Occurrence (%)
Aspergillus niger
198
30.18
Aspergillus flavus
97
14.79
Aspergillus fumigatus
5
0.76
Aspergillus terreus
28
4.30
Fusarium oxysporum
162
24.70
Fusarium compacticum
26
3.96
Penicillium oxalicum
117
17.80
Penicillium chrysogenum
8
1.22
Fusarium proliferum
8
1.22
Aspergillus tamari
7
1.07
Total
656
100

F.
P.
A.
A.
A.
A.
A.
F.
F.
P.
%
Ogundijo and Adetunji
Table 2.Occurence of each fungal organism in the different study locations
Locations
UI
0
0
26
0
9
0
4
32
0
18
13.6
Olunloyo
7
0
36
0
13
2
0
23
14
17
17.1
Oja Oba
0
0
35
0
12
0
9
33
0
29
18.0
Olorunsogo
0
0
36
6
26
0
8
29
2
7
17.4
Akinyele
1
0
29
1
11
1
4
31
6
15
15.1
Olodo
0
8
36
0
26
2
3
14
4
31
18.9
Total
8
8
198
7
97
5
28
162
26
117
Table 3. Fungal loads for sampled locations.
Study locations Location Type Sample size
(n)
Total fungal count +SD
(Log cfuml
-1
)
UI slaughter house Abattoir 40 4.14 ± 1.29
Olunloyo market Meat Market 70 4.73 ± 0.64
Oja Oba market Meat Market 60 4.38 ± 1.20
Olorunsogo market Abattoir/Meat Market 100 4.55 ± 0.88
Akinyele slaughter house Abattoir 40 4.26 ± 1.18
Olodo market Meat Market 50 4.68 ± 0.18
Mean (Log cfuml-1) 4.46 ± 0.24
n = 360
Discussion
The mean total fungal counts for the study
locations of 4.46 logCFUml-1 as reported
in this study exceeded the FAO/WHO
standard limit of 2.0 logCFUml-1 for total
microbial count offood products and water
(FAO/WHO, 2000). Penicillium oxalicum,
A. flavus, A. niger, A. terreus, and A. tamari
among other isolates were also reported by
Ehigiator et al. (2014), which were isolated
from shrimps in local meat shops in Benin,
except for A. fumigatus. This may be due to
the difference in the nature of samples
collected. Penicillium oxalicum was
similarly isolated in the work of Fasanmi et
al. (2010) and Ehigiator et al. (2014).
Fasanmi et al. (2010) also reported the
identification of Saccharromyces spp.
which was absent for this study due to the
initial precaution taken to prevent yeast
contamination of the cultured plates. A.
flavus, A. fumigatus and A. terreus as
reported in this study were not observed in
the study of Fasanmi et al. (2010). This
could be due to the difference in the studied
locations and sample size (50 samples as

Ibadan Journal of Agricultural Research Vol. 14(1), 2018
compared to 360 samples for this study).
According to Anand and Tiwary (2010),
Aspergillus flavus is the second leading cause
of invasive and non-invasive aspergillosis. The
isolation of A. fumigatus and A. flavus in
food processing areas of Olunloyo meat
market, Olodo meat market and Akinyele
slaughter house poses public health risk to
the consumers and meat sellers if proactive
measures are not put into place as
appropriate.
Penicillium spp. and Aspergillus spp.
as isolated from this study also confirmed
the ubiquitousness of fungi species through
similar isolation of these fungal organisms
together with Cladosporium spp. and yeasts
from both kitchen and other facilities in
school environment as carried out by
Shelton et al. (2002) and Lignell, (2008).
Bryden, (2007) identified Aspergillus,
Fusarium and Penicillium species as
mycotoxin producers, while Hymery et al.,
(2014) further defined them as food
spoilage fungi. Various species of
Fusarium have been known to produce
mycotoxins, which have osteogenous action
and significantly toxic to the reproductive
system of animals and humans, according
to Milicevic et al., (2010). The most
important of these mycotoxin-producers are
Aspergillus species as they are responsible
for the production of aflatoxins (Bennet and
Klich, 2003). A. flavus has been identified
as the most potent producer of aflatoxins
with mutagenic, hepatotoxic, and
carcinogenic properties (Zhang et al., 2012)
which have been confirmed to have
detrimental effects on both humans and
animals (Barret, 2000). Mohamed (2010)
also described the economic importance of
mycotoxins in animal production.
Therefore, the presence of A. flavus and
Fusarium species from meat-processing
areas in this study poses a threat to public
health, as supported by Pitt, (2000) and
Hymery et al. (2014), in meat markets and
slaughter houses in Ibadan.
According to Abdullahi et al. (2006),
unhygienic practices in abattoirs around the
meat markets are associated with potential
health risk to consumers due to the
presence of pathogens in meat and
environmental contamination. Also, lack or
inadequate veterinary inspection as
observed in most of the abattoirs and meat
markets visited, supplemented with lack of
potable water, proper waste disposal
facilities, and sanitary inspectors, is now
becoming a normal trend in many
slaughterhouses in Nigeria (Okoli et al.,
2006). These factors may contribute to the
high fungal contamination observed in this
study. Increased level of fungal
contamination in food processing
environment is expected in time as a result
of the biofilm forming abilities of
Aspergillus organisms as reported by
Ogundijo and Adetunji (2017).
Conclusion and Recommendation
There is strong compromise in food safety
with fungal contamination in the meat
processing areas covered in this study.
More research focus should be driven
towards preventing an outbreak or
emergence of fungal zoonoses in the
studied locations and other related areas.
Thisis attainable by ensuring that strict
hygiene and sanitation measures be put in
place in meat markets and abattoirs, in
order to ensure cleanliness and safety of
meat tables and slaughter slabs
respectively, to ensure meats and meat
products are safe and wholesome for
consumption.

Ogundijo and Adetunji
Acknowledgements
This work was sponsored in part by the
2010 Senate Research Grant (SRG) of the
University of Ibadan, Ibadan, Nigeria.
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