Salmonella in native “village” chickens (Gallus domesticus): prevalence and

risk factors from farms in South-Central Peninsular Malaysia

Saleh Mohammed Jajere, Latiffah Hassan,1 Saleha Abdul Aziz, Zunita Zakaria, Jalila Abu,
Fauziah Nordin, and Nik M. Faiz

Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan,
Malaysia

ABSTRACT Village chicken or Ayam Kampung, vidual bird-level was 2.5% (17/675, 95% CI: 1.6 to 4.0).
common to Southeast Asian countries, has always All eggs screened were negative; in the case of envi-
been regarded as superior in comparison to commer- ronmental samples, however, Salmonella was detected
cial broiler chicken in terms of wholesomeness and in 5.14% (9/175), 7.14% (5/70), and 5.0% (3/60) for
health benefits. The current study investigates the water, feed, and flies, respectively. A total of 34 iso-
prevalence and risk factors of Salmonella among vil- lates and 8 Salmonella serotypes were identified. Wel-
lage chicken flocks from the central and southern states tevreden (20.6%) was the most common, followed by
of Peninsular Malaysia. A total of 35 village flocks were Typhimurium and Agona (17.6%), Albany and Enteri-
sampled from Selangor (n = 19), Melaka (n = 10), tidis (8.8%), Molade (5.9%), Corvallis and Schleissheim
Johor (n = 4), and Negeri Sembilan (n = 2). In to- (2.9%), and others grouped as Salmonella spp. (11.8%).
tal, 1,042 samples were collected; these included cloacal Multivariable logistic regression models revealed that
swabs (n = 675), eggs (n = 62), pooled drinking wa- Salmonella positivity among flocks could be strongly
ter (n = 175), pooled feeds (n = 70), and pooled flies predicted by storage of feeds (uncovered feeds; OR =
(n = 60). Isolation of Salmonella from cloacal swabs, 10.38; 95% CI: 1.25 to 86.39; p = 0.030) and uncovered
poultry drinking water, and feeds was carried out ac- water tanks (uncovered tank; OR = 6.43; 95% CI: 1.02
cording to the protocols and recommendations of the to 40.60; p = 0.048). The presence of Salmonella in vil-
World Organization for Animal Health (OIE) terres- lage chickens in the study area was lower than that of
trial manual. The prevalence of Salmonella at an indi- commercial chickens in Malaysia.
Key words: village chicken, Salmonella, risk factor, prevalence, Peninsular Malaysia
2019 Poultry Science 98:5961–5970
http://dx.doi.org/10.3382/ps/pez392

INTRODUCTION for local households (Aini, 1990; Padhi, 2016). These

chickens are free to roam and scavenge for food, and
Native “village” chicken or Ayam Kampung is defined are fed leftovers and other household scraps to supple-
as a hybrid, the result of natural cross-breeding between ment their dietary feed requirements. However, since
the Malay fowl, jungle fowl, and mixed exotic breeds the emergence of highly pathogenic avian influenza,
brought in during periods of European colonization the traditional practice of rearing small flocks of free-
(Azahan and Zahari, 1983; Azahan, 1994). The plumage range chickens has been largely discouraged (Safman,
color of village chickens varies considerably with the 2010).
most common being a black-red variety (Azahan et al., Village chicken is common to many Southeast Asian
1980; Azahan and Zahari, 1983; Azahan, 1994). In countries, including Malaysia, and has always been re-
Southeast Asia and other developing countries, village garded as superior in comparison to commercial broiler
chickens are mainly raised in the backyard, variously chicken in terms of wholesomeness and health benefits
providing both a side income and source of protein (Aini, 1990; Hassan et al., 2005). These chickens are
customarily raised with little to no antibiotics or other
© The Author(s) 2019. Published by Oxford University Press on be- drugs. In recent years, rising awareness of issues per-
half of Poultry Science Association. This is an Open Access article dis-
tributed under the terms of the Creative Commons Attribution Non-
taining to animal welfare and the drugs used in com-
Commercial License (http://creativecommons.org/licenses/by-nc/4. mercial poultry production (i.e., intensive production)
0/), which permits non-commercial re-use, distribution, and reproduc- has resulted in a rapid increase in the demand for free-
tion in any medium, provided the original work is properly cited. For range native chickens, which consumers considered to
commercial re-use, please contact [email protected].
Received April 26, 2019.
be safer and more wholesome (Hassan et al., 2005; Miao
Accepted June 20, 2019. et al., 2005; Rahman and Haziqah, 2015). Therefore
1 Corresponding author: [email protected]
Ayam Kampung products cover a larger niche market

5961
5962 JAJERE ET AL.

as compared to previous years because of emergent food ing details of all village chicken farmers. Village chicken
safety and animal welfare concerns. farms were randomly selected from each of the 4 study
Worldwide, Salmonella is the most common pathogen sites using Microsoft Excel 2011. In total, 35 village
causing foodborne illnesses, with most infections linked chicken flocks were selected and visited from Selangor
to the consumption of poultry or poultry products (n = 19), Melaka (n = 10), Johor (n = 4), and Negeri
(Mezal et al., 2014; Saravanan et al., 2015; Wang et al., Sembilan (n = 2) during the study period. The major-
2015). The presence of Salmonella in commercial poul- ity of farms investigated in this study were from Se-
try, its meat, and poultry products has been well stud- langor because Selangor is home to the largest num-
ied (Rusul et al., 1996; Najwa et al., 2015; Thung et al., ber of village flocks as compared to other states in this
2016). Moreover, several studies from across the globe study.
have studied the risk factors associated with Salmonella
contamination in broiler chickens (Rose et al., 1999; Study Design
Arsenault et al., 2007; Marin et al., 2011). The reported
prevalence of Salmonella in live commercial poultry in Selected farms were contacted via telephone and an
Malaysia was reported at 14.9% (n = 1911) (Ong et al., appointment to visit the farm was set. A cross-sectional
2014). However, little information is available with study was employed whereby all samples, as well as data
respect to native chickens. Native chickens generally on farm management and production systems, were col-
have considerably greater exposure to environmental lected during the same visit. Farm management systems
elements due to having been free-ranged. In addition, were categorized as either free-range, semi-intensive,
native chickens are not subjected to selective breeding or intensive. For free-range, birds are let free to roam
aimed at producing pathogen-free or disease-free flocks. about and scavenge, fed leftovers and other household
Therefore, native chickens may represent a greater risk scraps, provided with little or no man-made shed, and
for Salmonella infection. The aim of this study is to roost in the evening in a common area. This is the most
determine the prevalence of Salmonella among the common system used in resource-poor village house-
local native chickens, and to identify those factors that holds (Lawal et al., 2016); consequently, most native
play a role in the perpetuation of the pathogen in these chickens are raised according to this management sys-
flocks. tem. For semi-intensive, birds are housed during the
night and released the following morning to scavenge.
MATERIALS AND METHODS The house can vary from crude wire-meshed to more
sophisticated structures. We found a sizeable number
Study Area of village chicken farmers to be practicing this sys-
tem. While little attention is given to chicken vaccina-
Malaysia is located north of the equator, and is bor- tion or other treatments in the case of both free-range
dered to the north by Thailand and in the south by and semi-intensive system, intensively farmed birds are
Singapore. Malaysia is a country divided by the South housed throughout their life cycle and undergo a basic
China Sea, with Peninsular Malaysia to the west, and scheduled of preventive measures and vaccinations to
East Malaysia, comprising Sabah and Sarawak, to the guard against poultry and other diseases. Other good
east. Peninsular Malaysia is made up of 11 states. This farm management practices, such as all-in-all-out, may
study sampled farms from 4 states, namely Selangor, also be practiced in intensive production systems. The
Johor, Melaka, and Negeri Sembilan in the central and housing type varies from cages to more sophisticated
southern regions of the country. These states have a cu- housing as seen in large commercial production sys-
mulative 220 village chicken farms, comprising approx- tems. In general, this system is the least common for
imately 24.86% (n = 885) of the total recorded village raising village chickens in the region. However, with the
chicken farms in Peninsular Malaysia. increasing interest and demand for village chicken prod-
ucts, this type of production system is fast becoming
Sample Size more prevalent.

The sample size was calculated using Epi info statis- Sample Collection from Village Flocks
tical software (Atlanta, GA). The calculation was based
on the assumption of an expected prevalence of 14.9% Sample collection was performed between November
and 3.1%, respectively, for cloacal swabs and eggs (Ong 2016 and February 2018. Cloacal swabs were collected
et al., 2014). With a desired absolute precision of 5%, using sterile swab sticks (Oxoid, Cheshire, England),
95% level of confidence, and a large population size, and immersed in 10 mL buffered peptone water (BPW,
sample sizes of at least n = 235 and n = 56 were Oxoid). Pooled feed samples were collected from feeders
required for cloacal swabs and eggs, respectively. The located within the poultry pens using sterile polythene
study population comprises village chickens from cen- bags. Five 10 mL samples of pooled poultry drinking
tral and southern states in Peninsular Malaysia. These water were collected using 10 mL sterile syringes from
states were selected based on a sampling frame ob- drinkers. Eggs were purchased whenever available at
tained from the Ayam Kampung Association compris- the farms and packed using sterile polythene bags. All
 SALMONELLA IN NATIVE “VILLAGE” CHICKENS 5963
samples collected during visitation were immediately incubated at 37°C for 18 to 24 h. Spherical transparent
transported in an ice-cooled box to the Veterinary Pub- red halo colonies with typical black centers on XLD, and
lic Health Laboratory at the Faculty of Veterinary moist spherical red/pink colored colonies with redden-
Medicine, Universiti Putra Malaysia, for further pro- ing of the media on BGA plates, were selected as pre-
cessing or kept in an isolated refrigerator for sample sumptive Salmonella colonies and further sub-cultured
storage until processed the following day. Flies were on nutrient agar plates (NA, Oxoid) and incubated at
collected from the study sites as described elsewhere 37°C for 18 to 24 h. A total of 3 suspected colonies each
(Choo et al., 2011). from XLD and BGA plates (positive) were picked for
sub-culturing on NA agar plates. Following incubation
Isolation of Salmonella of the NA agar plates, triplicate suspected colonies were
subjected to serological tests and further biochemical
Cloacal Swabs, Flies, Poultry Drinking Water, characterization as per the standard methods (Choo
and Feeds Isolation of Salmonella from cloacal swabs, et al., 2011).
poultry drinking water, and feeds was carried out Serological and Biochemical Confirmation of
according to the protocols and recommendations of Suspected Salmonella Colonies For serological
the OIE terrestrial manual (www.oie.int) and ISO identification, Polyvalent Antiserum “O” was used ac-
6579:2002. Briefly, for isolation from cloacal swabs, the cording to the manufacturer’s instructions. Biochemi-
swab sticks immersed in 10 mL BPW (Oxoid) were in- cal confirmation of the presumptive Salmonella isolates
cubated at 37°C for 18 to 24 h. For isolation from flies, was done as described elsewhere (Choo et al., 2011).
trapped flies were processed as previously described Interpretations followed standard recommended guide-
(Choo et al., 2011), immersed in 10 mL BPW (Oxoid), lines (Mikoleit, 2014). Confirmation to serogroup level
then incubated at 37°C for 18 to 24 h. For isolation was by poly “O” and poly “H” (Phase 1 and Phase 2)
from drinking water, 10 mL of the water collected from antiserum. All isolates were sent to the Veterinary Re-
the farms were each added to 90 mL of BPW and sub- search Institute in Ipoh (Peninsular Malaysia) for fur-
sequently incubated at 37°C for 18 to 24 h. Similarly, ther confirmation and serotyping. A flock was defined
the feeds collected from the farms were first weighed to as Salmonella-positive if at least 1 cloacal sample was
10 g using weighing balance, after which the 10 g of feed positive regardless of the positivity of other environ-
was inoculated into 90 mL of BPW (Oxoid) and incu- mental samples from the same flock.
bated at 37°C for 18 to 24 h. All samples were further
processed for Salmonella identification as described Data Collection
below.
Isolation of Salmonella from the Eggshell Sur- A questionnaire pertaining to village chicken pro-
face and Egg Yolk All broken or cracked egg samples duction was developed and piloted with a sample of
were discarded. Eggshell surface was swabbed using a village chicken farmers in the state of Selangor. Ques-
sterile cotton swab soaked in sterile normal saline so- tionnaire items were refined based on the feedback re-
lution. The swabbed stick was immersed into 10 mL ceived from farmers. Informed consent was obtained
Tryptic soy broth (TSB) (Oxoid) and incubated at 37°C from each farmer before being administered the ques-
for 24 h. For the egg yolk, isolation was done as recom- tionnaire. The questionnaire was written in both En-
mended by the US Food and Drug Administration Bac- glish and Bahasa Melayu languages. Information col-
teriological Analytical Manual, and by a previous study lected from the questionnaire included data on flock
(Zhang et al., 2013). Briefly, the egg sample surface was size, income level (Malaysian Ringgit), feed and wa-
disinfected by immersing whole eggs in a 3:1 disinfec- ter sources, storage of water and feed, poultry produc-
tant solution consisting of 3 parts 70% ethyl alcohol tion system, the presence of wild birds, and other farm
and 1 part iodine/potassium iodide solution for 10 s management practices, such as the management of sick
(Zhang et al., 2013). Surface-disinfected eggs were air- birds, the disposal of dead birds in the flock, etc.
dried at room temperature before eggshell was cracked.
Egg yolks were stirred in a stomacher for thorough mix- Data Analysis
ing. 10 mL of the mixed egg yolk content was then in-
oculated into 90 mL of TSB (Oxoid) and incubated at Data was entered and managed in Microsoft Office
37°C for 24 h. Excel 2011. Descriptive statistics were utilized to de-
Identification of Salmonella Following the initial termine the prevalence and proportions of Salmonella
pre-enrichment in BPW (Oxoid), and in TSB (Oxoid) positivity according to the independent variables. Chi-
in the case of egg samples, 0.1 mL of the pre-enriched square, Fisher’s exact test, or simple logistic regression
samples were added to 10 mL of Rappaport Vassil- was used for the univariable exploratory analysis to
iadis broth (RVB) (Oxoid) and incubated at 42°C for identify risk factors associated with the outcome vari-
24 h. RVB enriched samples were streaked onto 2 selec- able. Risk factors were analyzed by multivariable lo-
tive media, namely Brilliant Green Agar (BGA) supple- gistic regression models using forward Wald stepwise
mented with Novobiocin antibiotic (Oxoid), and Xylose regression on variables with p < 0.05 from the uni-
Lysine Desoxycholate (XLD) (Oxoid). Plates were then variable analysis. Risk of Salmonella infection amongst
5964 JAJERE ET AL.

Table 1. Description of village chicken farms in the central and southern regions of Peninsular
Malaysia.

Farm locations Number of farms Relative % Flock size1 Range1

Selangor 122 55.5 94,178 4–15,000
Negeri Sembilan 2 0.9 21,200 1,200–20,000
Melaka 85 38.6 25,911 20–4,000
Johor 11 5 563,000 8,000–100,000
Total 220 100 704,289 4–100,000
1
Number of village chickens raised.

Table 2. Salmonella from “Ayam kampung” farms in the central was 75%, 25%, and 40% for free-range (n = 8), semi-
and southern regions of Peninsular Malaysia (n = 35). intensive (n = 12), and intensive (n = 15) management
Sources Total Positive (%) system, respectively (Table 3). In total, 34 Salmonella
isolates were recovered: 18 isolates from cloacal swabs,
Cloacal swab 675 17 (2.75)
Eggs 62 00 9 from pooled poultry drinking water, 5 from pooled
Poultry drinking water1 175 9 (5.14) feed and 3 fly samples (Table 2). Of the 34 isolates, we
Feeds 70 5 (7.14) identified S. Weltevreden (n = 7), S. Agona (n = 6), S.
Flies2 60 3 (5.00)
Typhimurium (n = 6), S. Albany (n = 3), S. Enteritidis
Overall 1,042 34 (3.26) (n = 3), S. Molade (n = 2), S. Corvallis (n = 2), and
1
5 samples of water 10 mLs each was obtained from each farm. S. Schleissheim (n = 1). Other unidentified serotypes
2
2 pooled samples of 5 flies each was sampled from each farm. were grouped as Salmonella spp (n = 4) (Figure 1).

birds was expressed as an adjusted odds ratio (OR)

with 95% confidence interval. All analyses were per-
formed using SPSS version 22.0 (IBM, Armonk, NY)
at the significance level α = 0.05. Univariable Analysis of Risk Factors for
Salmonella
RESULTS Univariable analysis revealed risk factors associated
with salmonellosis in village chickens. These risk factors
Farm Demography and Distribution in the included sources of feeds (χ2 = 5.872; p = 0.053), farm
Central and Southern Peninsular Malaysia feed storage (χ2 = 9.343; p = 0.002), farm water storage
(χ2 = 4.644; p = 0.031), sick bird management practices
According to the sampling frame provided, there were
(χ2 = 6.076; p = 0.014), and the disposal of dead birds
222 farms located in central and southern Peninsular
from the farm (χ2 = 3.902; p = 0.048) (Table 3).
Malaysia (Table 1). Selangor had the highest number
of village chicken farms (n = 122), followed by Melaka
(n = 85), Johor (n = 11), and Negeri Sembilan (n = 2).
Although Johor has fewer village chicken farms, it has
the highest density of birds as compared to the other Multiple Logistic Regression of Risk
states combined. The majority of village chicken flocks Factors for Salmonella
in Selangor are backyard operations, with flock sizes
ranging from as low as 10 birds, to as high as several Multivariable regression revealed that uncovered feed
hundred. Majority of the farms we sampled followed storage (OR = 10.4; 95% CI: 1.25 to 86.39; p = 0.030)
intensive production system (n = 15, 43%), followed by and uncovered water storage (OR = 6.4; 95% CI: 1.02
semi-intensive (n = 12; 34%), and free-range (n = 8; to 40.6; p = 0.048) were significantly associated with
23%). an increased odds of Salmonella positivity (Table 4).
Village chickens in farms with uncovered feeds storage
Salmonella Prevalence and Serotypes were approximately 10 times more likely to test positive
for Salmonella as compared to those from farms with
Of the 35 farms screened, 15 farms were positive, covered storage. Likewise, village chickens from farms
giving a farm-level prevalence of 42.9% (95% CI: 28.0, with uncovered water storage were approximately 6
59.2). Of the 675 cloacal swabs sampled from all farms, times more likely to test positive for Salmonella as com-
17 (2.5%; 95% CI 1.6, 4.0) were positive for Salmonella pared to those from farms with covered water storage
(Table 2). All 62 eggs screened were negative. The (Table 4). However, while sick bird management prac-
Salmonella detection rates from pooled poultry drink- tices (i.e., not isolating sick birds from healthy birds;
ing water, feeds, and flies were 5.14%, 7.14%, and OR = 2.56; 95% CI: 0.40 to 16.22; p = 0.318) fitted the
5.0%, respectively (Table 2). According to the manage- final regression model, the result was not significant
ment practices, the percentage of Salmonella positive (Table 4).
 SALMONELLA IN NATIVE “VILLAGE” CHICKENS 5965
Table 3. Univariable analysis for risk factors associated with the occurrence of Salmonella amongst village
chickens from the central and southern Peninsular Malaysia.

Variables Frequency Positive (%) Chi-square (χ2) P-value

Flock size (number of birds in the farm)
<500 18 9 (50.0) 0.787 0.675
500–1,000 6 2 (33.3)
>1,000 11 4 (36.4)
Income level (Ringgit)
<500 16 7 (43.8) 0.048 0.976
500–1,200 9 4 (44.4)
>1,200 10 4 (40.0)
Level of education of the farmer
Primary school 2 0 3.241 0.356
Secondary 9 5 (55.6)
Certificate 9 5 (55.6)
University degree 15 5 (33.3)
Source of feeds in the farm1
Homemade 20 12 (60.0) 5.872 0.053
Commercially prepared 12 2 (16.7)
Table scraps 3 1 (33.3)
Storage of feeds in the farm
Covered 21 13 (61.9) 9.343 0.002
Uncovered 14 2 (14.3)
Use of additives/growth promoters
Yes 24 8 (33.3) 2.828 0.093
No 11 7 (63.6)
Source of water in the farm
Rain water 2 1 (50.0) 2.106 0.349
Well water 9 2 (22.2)
Tap/Borehole 24 12 (50.0)
Storage of water in the farm
Covered tank storage 16 10 (62.5) 4.644 0.031
Uncovered tank storage 19 5 (26.3)
Poultry production system
Free-range 8 6 (75.0) 4.988 0.083
Semi-intensive 12 3 (25.0)
Intensive 15 6 (40.0)
Frequency of poultry manure disposal
Disposed weekly 10 6 (60.0) 2.622 0.270
Disposed monthly 17 5 (29.4)
Never 8 4 (50.0)
Frequency of cleaning house litters
Weekly 9 1 (11.1) 5.213 0.074
Monthly 23 12 (52.2)
Never 3 2 (66.7)
Wild birds in the farm
Yes 21 11 (52.4) 1.944 0.163
No 14 4 (28.6)
Accessibility of the farm to the public
Presence of fence 27 12 (44.4) 0.122 0.727
No fence 8 3 (37.5)
Presence of other animals (Dogs, Cats, Goats) in the farm
Yes 21 9 (42.9) 0.000 1.000
No 14 6 (42.9)
Management of sick birds in the farm
Isolated from healthy birds 20 5 (25.0) 6.076 0.014
No isolation 15 10 (66.7)
Disposal of dead birds in the farm
Disposed inside farm premises 27 14 (51.9) 3.902 0.048
Disposed outside farm premises 8 1 (12.5)
Control of vermins in the farm
Yes 20 9 (45.0) 0.087 0.767
No 15 6 (40.0)
Use of disinfectants and footbaths
Yes 6 2 (33.3) 0.268 0.605
No 29 13 (44.8)
Use of antibiotics in the farm
Yes 18 10 (55.5) 2.440 0.118
No 17 5 (29.4)
5966 JAJERE ET AL.

Table 3. Continued

Variables Frequency Positive (%) Chi-square (χ2) P-value

Vaccination of birds in the farm
Yes 18 8 (44.4) 0.038 0.845
No 17 7 (41.2)
Veterinary consultancy services
Yes 19 7 (36.8) 1.703 0.427
No 16 8 (50.0)
Salmonella isolated from environmental samples2
Yes 10 (28.6) 1.680 0.266
No 7 (20.0)

1
Homemade poultry feeds are made by village farmers at home with organic ingredients and other raw materials from local feed stores.
2
Environmental samples comprised poultry drinking water, poultry feeds, and flies in and around the pens.

Figure 1. Distribution of the serotypes of Salmonella isolated from village chickens in Peninsular Malaysia.

Table 4. Multiple logistic regression of risk factors associated with the occurrence of salmonellosis
in village chickens from the central and southern Peninsular Malaysia.

95% confidence
Variables B S.E Wald df P-value Adjusted OR1 interval
Storage of feeds 2.341 1.081 4.690 1 0.030 10.387 1.25−86.39
(Uncovered)
Storage of water 1.860 0.941 3.911 1 0.048 6.425 1.02−40.60
(Uncovered tank storage)
Management of sick birds 0.940 0.942 0.995 1 0.318 2.560 0.40−16.22
(Number isolation of sick birds)

1
OR, Odds ratio.

DISCUSSION mercial production may explain these observed differ-

ences. It has been reported that overcrowding and high-
Village chickens and their products have been gain- density population increases Salmonella transmission
ing in popularity in Malaysia because of their presumed among commercially-raised poultry despite preventa-
wholesomeness and safety. This study found that al- tive measures (Bailey, 1988; Huneau-Salaün et al., 2009;
though the flock-level prevalence for Salmonella was Foley et al., 2011; Im et al., 2015; Ansari et al., 2017).
quite high, chicken-level prevalence was much lower at Moreover, high-density production leads to stress, thus
2.5% (n = 675). This prevalence is much lower than lowering individual ability to resist infection (Gomes
that previously published by Ong et al. for local com- et al., 2014; Gast et al., 2017).
mercial chickens—14.9% (n = 1911) in poultry farms Compared to other studies of Salmonella in back-
(Ong et al., 2014). This finding was unexpected because yard chicken, we found higher Salmonella levels than a
village chickens are raised with few structured disease South Australian that reported an estimated animal-
controls or Salmonella control programs. Moreover, the level prevalence of 0.02% after screening (n = 115)
farms have minimum or non-existent biosecurity mea- pooled cloacal swab samples from 30 backyard flocks
sures, thus exposing chickens to various environmental (Manning et al., 2015). However, our estimate is lower
elements before they attain marketable weight. How- than findings reported from West Bengal in India (15%;
ever, we believe that overcrowding and stress in com- n = 40, Samanta et al., 2014), and from Iran (5.8%;
 SALMONELLA IN NATIVE “VILLAGE” CHICKENS 5967
n = 85) following testing of 422 cloacal swab samples confirmed Salmonella enterica serovar Weltevreden as
from 35 backyard flocks (Jafari et al., 2007). Our find- the causal agent of the foodborne infection. On the
ings are in fact similar to those in Paraguay (3.5%; other hand, S. Agona is among the most common
n = 400) of 50 backyard flocks (Leotta et al., 2010), non-typhoidal serovars of Salmonella isolated from hu-
and the central region of Saudi Arabia (2.2%) obtained mans in Europe, and was responsible for the 2008 pan-
from broiler parent flocks (Saad et al., 2007). Notwith- European outbreak (McCusker et al., 2014).
standing, the aforementioned comparisons should be re- In this study, we recorded 5.14%, 7.14%, and 5.0%
garded with caution given various differences in the Salmonella in samples of drinking water, feeds, and
techniques used for isolation, limited sampling areas flies, respectively (Table 2). The isolation rates of 5.14%
(as seen in the study from Paraguay, which used a rel- from drinking water and 7.14% from poultry feeds were
atively small sample size, and the study from Iran), lower than the 36% reported in commercial poultry
and differences in the population dynamics of domi- drinking water in Thailand (Sasipreeyajan et al., 1996),
nant Salmonella serovars in each country. Other au- and 22.2% reported from 36 bulk commercial poultry
thors (e.g., Carrique-Mas and Davies, 2008) have ex- feeds in Nigeria (Okoli et al., 2006). However, the iso-
plained that the prevalence of Salmonella measured in lation rates reported in our study were also slightly
birds using cloacal swabs is often low because of, among higher than 3.3% (poultry water) and 2.5% (poultry
other factors, the intermittent shedding of the organ- feeds) as reported from layer farms in Northern In-
ism and relatively low number of organisms excreted dia (Singh et al., 2013). Contaminated poultry feed
by infected birds. In our study, more than half of the and drinking water are critical sources of Salmonella
farmers surveyed during the study stated that they used infection and have been associated with transmission
antibiotics when their birds are sick, which could have and the perpetuation of Salmonella in poultry farms
probably reduce the recovery rate of Salmonella in our (Frederick and Huda, 2011). Although we did not
study. In addition, diverse age range of sampled chick- find a significant association of Salmonella being
ens could also be a contributing factor to the differ- present in the environmental samples, to the farm-level
ences observed. Moreover, lack of further enrichment Salmonella prevalence in this study, several previous
step with either selenite/tetrathionate broth after the studies highlight the importance of environmental con-
pre-enrichment with buffered peptone water could be tamination (Sasipreeyajan et al., 1996; Okoli et al.,
another reason for the lower isolation rate observed in 2006; Singh et al., 2013; Ong et al., 2014; Samanta et al.,
this study. 2014).
We recovered 8 serotypes of Salmonella enterica, Other studies have reported Salmonella in eggs
with a predominance of Weltevreden, Agona, and (Assefa et al., 2011; Murakami et al., 2013; Singh et al.,
Typhimurium. S. Typhimurium—serotypes with well- 2013; Ayachi et al., 2015); however, our study did
established histories of causing various human infec- not. This finding is consistent with the low levels of
tions, and frequently isolated among clinical cases in Salmonella infection found among village flocks. Nev-
Malaysia (Ministry of Health, Malaysia, 2015; Karim ertheless, we screened only 62 eggs from the flocks in
et al., 2017). One recent study reported that S. Ty- this study, which may be too small to detect Salmonella
phimurium was among the most commonly associated in the eggs.
non-typhoidal Salmonella poultry-related salmonellosis Uncovered storage of both poultry drinking water
in the United States (Shah et al., 2016). Previous and feeds allows complete access to rodents, flies, and
studies have not reported the other 2 aforementioned the droppings of wild bird. Rodents and wild birds
serotypes to be as predominant in ready-to-eat foods, are known Salmonella carriers (Andrés-Barranco et al.,
vegetables, beef, raw meat, carcasses, or live birds 2014; Mustaffa et al., 2014; Matias et al., 2016; Ribas
in Malaysia (Rusul et al., 1996; Ong et al., 2014; et al., 2016). A number of studies have highlighted the
Najwa et al., 2015; Nidaullah et al., 2017). Wel- role of flies, rodents, and wild birds in the transmission
tevreden and Agona serotypes are pathogenic and and perpetuation of Salmonella infection among poul-
have caused multiple foodborne disease outbreaks in try flocks and other farm animals. Rodents can carry
Malaysia (Ministry of Health, Malaysia, 2015; Karim Salmonella in their intestinal tracts asymptomatically
et al., 2017), and elsewhere (Centers for Disease (Zamora-Sanabria and Alvarado, 2017), and are the re-
Control and Prevention, 2006; Bruun et al., 2009; ported vectors and amplifiers of Salmonella in farm an-
Whelan et al., 2010; Russo et al., 2013; Mba-Jonas imals (Meerburg and Kijlstra, 2007). Rodent droppings
et al., 2018). For instance, an earlier study based on can remain a source of Salmonella contamination for
clinical records sourced from the Institute of Medi- up to 3 mo following infection with the feaces of sick
cal Research between 1983 and 1992 reported that animals (Davies and Wray, 1995; Zamora-Sanabria and
serotype Weltevreden was among the most predomi- Alvarado, 2017). On the other hand, flies also act as
nant Salmonella isolated from clinical cases in humans mechanical vectors, aiding transmission of the bacteria
(Yasin et al., 1997). Recently, Malaysia’s Ministry of from one farm to another. Heavy fly populations have
Health reported on a foodborne outbreak associated been identified as a risk factor for Salmonella in poultry,
with a local dish called—laksa—that resulted in 2 dairy cattle, swine, and feedlot cattle (Vanselow et al.,
deaths. The laboratory results from the clinical samples 2007). Wild birds and other wildlife can introduce and
5968 JAJERE ET AL.

disseminate the bacteria via the contamination of feed, Ayachi, A., O. Bennoune, N. Heleili, and N. Alloui. 2015. Minor
water, or direct environmental contamination (Zamora- Salmonella: potential pathogens in eggs in Algeria. J. Infect. Dev.
Ctries. 9:1156.
Sanabria and Alvarado, 2017). Azahan, E. A. E. 1994. The red and black-red native chickens
of Malaysia. Malaysian Agric. Res. Dev. Inst. Res. J 22:73–
CONCLUSION 78.
Azahan, E. A. E., C. P. Seet, and O. Zainab. 1980. A comparative
study of physiological and productive performance of local (Kam-
We observed a lower animal level of prevalence of pung) and commercial chickens. Malaysian Agric. Res. Dev. Inst.
Salmonella infection among village chickens compared Res. J. 52:61–70.
to reported prevalence among commercial broiler chick- Azahan, E. A. E., and W. M. Zahari. 1983. Observation on some
ens. Risk factors for infection include uncovered wa- characteristics of carcass and meat of Malaysian kampong chick-
ens. Mardi Res. Bull. 11:225–232.
ter and feed storage. These practices allow access of Bailey, J. S. 1988. Integrated colonization control of Salmonella in
poultry feeds and drinking water to flies, rodents, and poultry. Poult. Sci. 67:928–932.
wild bird droppings. To improve the safety of village Bruun, T., G. Sørensen, L. P. Forshell, T. Jensen, K. Nygard,
chicken, farmers should be advised to undertake sim- G. Kapperud, B. A. Lindstedt, T. Berglund, A. Wingstrand,
R. F. Petersen, L. Müller, C. Kjelsø, S. Ivarsson, M.
ple good management practices, such as storing feed Hjertqvist, S. Löfdahl, and S. Ethelberg. 2009. An out-
and water in covered containers, and improving biose- break of Salmonella Typhimurium infections in Denmark,
curity to reduce the accessibility of wild birds and Norway and Sweden, 2008. Euro Surveill 14. PMID: 19317986.
animals. https://www.eurosurveillance.org/content/10.2807/ese.14.10.
19147-en#html_fulltext.
Carrique-Mas, J. J., and R. H. Davies. 2008. Sampling and bacteri-
ological detection of Salmonella in poultry and poultry premises:
a review. Rev. Sci. Tech. OIE 27:665–677.
ACKNOWLEDGMENTS Centers for Disease Control and Prevention. 2006. Multistate out-
break of Salmonella typhimurium infections associated with eat-
We thank the local Ayam Kampung Association of ing ground beef−United States, 2004. MMWR Morb. Mortal
Peninsular Malaysia and farmers for their kind support Wkly. Re. 55:180–218.
and collaboration. Choo, L. C., A. A. Saleha, S. S. Wai, and N. Fauziah. 2011. Iso-
lation of Campylobacter and Salmonella from houseflies (Musca
domestica) in a university campus and a poultry farm in Selangor,
FUNDING Malaysia. Trop. Biomed. 28:16–20.
Davies, R., and C. Wray. 1995. Mice as carriers of Salmonella enter-
This research project was part of a doctoral study itidis on persistently infected poultry units. Vet. Rec. 137:337–
undertaken in the Faculty of Veterinary Medicine, Uni- 341.
Foley, S. L., R. Nayak, I. B. Hanning, T. J. Johnson, J. Han, and
versiti Putra Malaysia, and was funded by the UPM S. C. Ricke. 2011. Population dynamics of Salmonella enterica
Research Grant number 9522900. The funding source serotypes in commercial egg and poultry production. Appl. Env-
did not play any role in the conduct of the research, iron. Microbiol. 77:4273–4279.
nor in the writing or submission of the manuscript for Frederick, A., and N. Huda. 2011. Salmonellas, poultry house en-
vironments and feeds: a review. J. Anim. Vet. Adv. 10:679–
publication. 685.
Gast, R. K., R. Guraya, D. R. Jones, K. E. Anderson, and
D. M. Karcher. 2017. Frequency and duration of fecal shedding
COMPETING INTERESTS of Salmonella enteritidis by experimentally infected laying hens
housed in enriched colony cages at different stocking densities.
None declared. Front. Vet. Sci. 4:47.
Gomes, A. V. S., W. M. Quinteiro-Filho, A. Ribeiro, V. Ferraz-
de-Paula, M. L. Pinheiro, E. Baskeville, A. T. Akamine, C. S.
REFERENCES Astolfi-Ferreira, A. J. P. Ferreira, and J. Palermo-Neto. 2014.
Overcrowding stress decreases macrophage activity and increases
Aini, I. 1990. Indigenous chicken production in south-east Asia. Salmonella Enteritidis invasion in broiler chickens. Avian Pathol.
Worlds Poult. Sci. J. 46:51–57. 43:82–90.
Andrés-Barranco, S., J. P. Vico, V. Garrido, S. Samper, S. Herrera- Hassan, L., S. Z. Suhaimi, and A. A. Saleha. 2005. The detection and
León, C. de Frutos, and R. C. Mainar-Jaime. 2014. Role comparison of antimicrobial resistance pattern of vancomycin-
of wild bird and rodents in the epidemiology of subclinical resistant enterococci and Salmonella isolated from eggs of com-
Salmonellosis in finishing pigs. Foodborne Pathog. Dis. 11:689– mercial layers and free-range chickens. J. Vet. Malaysia 17:7–
697. 11.
Ansari, F., H. Pourjafar, S. Bokaie, S. M. Peighambari, M. Huneau-Salaün, A., C. Marianne, L. B. Sophie, L. Françoise, P.
Mahmoudi, M. H. Fallah, F. Tehrani, A. Rajab, S. A. Ghafouri, Isabelle, R. Sandra, M. Virginie, F. Philippe, and R. Nicolas.
and M. Shabani. 2017. Association between poultry density and 2009. Risk factors for Salmonella enterica subsp. enterica contam-
Salmonella infection in commercial laying flocks in Iran using a ination in 519 French laying hen flocks at the end of the laying
kernel density. Pak. Vet. J. 37:299–304. period. Prev. Vet. Med. 89:51–58.
Arsenault, J., A. Letellier, S. Quessy, V. Normand, and M. Im, M. C., S. J. Jeong, Y.-K. Kwon, O.-M. Jeong, M.-S. Kang, and
Boulianne. 2007. Prevalence and risk factors for Salmonella spp. Y. J. Lee. 2015. Prevalence and characteristics of Salmonella
and Campylobacter spp. caecal colonization in broiler chicken and spp. isolated from commercial layer farms in Korea. Poult. Sci.
turkey flocks slaughtered in Quebec, Canada. Prev. Vet. Med. 94:1691–1698.
81:250–264. Jafari, R. A., M. Ghorbanpour, and A. Jaideri. 2007. An investiga-
Assefa, M., A. Teklu, and H. Negussie. 2011. The prevalence tion into Salmonella infection status in backyard chickens in Iran.
and public health importance of Salmonella from chicken ta- Int. J. Poult. Sci. 6:227–229.
ble eggs, Ethiopia. Am. Eurasian J. Agric. Environ. Sci. 11:512– Karim, B. A., A. L. Latip, A. S. A. Shukor, N. A. Rashid, W.
518. M. W. Mohd, and F. Kamaludin. 2017. A large common source
 SALMONELLA IN NATIVE “VILLAGE” CHICKENS 5969
outbreak of Salmonella typhimurium linked to Kuala Terengganu Padhi, M. K. 2016. Importance of indigenous breeds of chicken for
night markets, Malaysia, 2014. OSIR J. 10:1–7. rural economy and their improvements for higher production per-
Lawal, J. R., S. M. Jajere, U. I. Ibrahim, Y. A. Geidam, I. A. Gulani, formance. Scientifica 2016:1–9.
G. Musa, and B. U. Ibekwe. 2016. Prevalence of coccidiosis among Rahman, W. A., and F. Haziqah. 2015. Ectoparasitic fauna of scav-
village and exotic breed of chickens in Maiduguri, Nigeria, Vet. enging chickens (Gallus domesticus) from Penang Island, Penin-
World 9:653–659. sular Malaysia. Malaysian J. Vet. Res. 6:33–42.
Leotta, G., K. Suzuki, F. L. Alvarez, L. Nuñez, M. G. Silva, L. Ribas, A., W. Saijuntha, T. Agatsuma, V. Prantlová, and S.
Castro, M. L. Faccioli, N. Zarate, N. Weiler, M. Alvarez, and J. Poonlaphdecha. 2016. Rodents as a source of Salmonella contam-
Copes. 2010. Prevalence of Salmonella Spp. in backyard chickens ination in wet markets in Thailand. Vector Borne Zoonotic Dis.
in Paraguay. Int. J. Poult. Sci. 9:533–536. 16:537–540.
Manning, J., V. Gole, and K. Chousalkar. 2015. Screening for Rose, N., F. Beaudeau, P. Drouin, J. Y. Toux, V. Rose, and P. Colin.
Salmonella in backyard chickens. Prev. Vet. Med. 120:241– 1999. Risk factors for Salmonella enterica subsp. enterica contam-
245. ination in French broiler-chicken flocks at the end of the rearing
Marin, C., S. Balasch, S. Vega, and M. Lainez. 2011. Sources of period. Prev. Vet. Med. 39:265–277.
Salmonella contamination during broiler production in Eastern Russo, E. T., G. Biggerstaff, R. M. Hoekstra, S. Meyer, N.
Spain. Prev. Vet. Med. 98:39–45. Patel, B. Miller, and R. Quick, and The Salmonella Agona Out-
Matias, C. A. R., I. A. Pereira, E. M. F. dos Reis, D. dos P. break Investigation Team. 2013. A recurrent, multistate out-
Rodrigues, and S. Siciliano. 2016. Frequency of zoonotic bacte- break of Salmonella serotype agona infections associated with dry,
ria among illegally traded wild birds in Rio de Janeiro. Braz. J. unsweetened cereal consumption, United States, 2008. J. Food
Microbiol. 47:882–888. Prot. 76:227–230.
Mba-Jonas, A., W. Culpepper, T. Hill, V. Cantu, J. Loera, J. Rusul, G., J. Khair, S. Radu, C. T. Cheah, and R. M. Yassin. 1996.
Borders, L. Saathoff-Huber, J. Nsubuga, I. Zambrana, S. Dalton, Prevalence of Salmonella in broilers at retail outlets, processing
I. Williams, and K. P. Neil. 2018. A multistate outbreak of human plants and farms in Malaysia. Int. J. Food Microbiol. 33:183–
Salmonella agona infections associated with consumption of fresh, 194.
whole papayas imported from Mexico—United States, 2011. Clin. Saad, A. M., D. M. Almujali, S. H. Babiker, M. A. M. Suhaib, K. A.
Infect. Dis. 66:1756–1761. Adelgadir, and Y. A. Alfadul. 2007. Prevalence of Salmonellae in
McCusker, M. P., K. Hokamp, J. F. Buckley, P. G. Wall, M. Martins, broiler chicken carcasses and poultry farms in the central region
and S. Fanning. 2014. Complete genome sequence of Salmonella of KSA. J. Anim. Vet. Adv. 6:164–167.
enterica serovar agona pulsed-field type SAGOXB.0066, cause of Safman, R. M. 2010. Avian influenza control in Thailand: balanc-
a 2008 pan-European outbreak. Genome Announc. 2:e01219–13, ing the interests of different poultry producers. Pages 169–206
2/1/e01219-13. in Avian Influenza: Science, Policy and Politics. I. Scoones, ed.
Meerburg, B. G., and A. Kijlstra. 2007. Role of rodents in trans- Earthscan, London, UK.
mission ofSalmonella andCampylobacter. J. Sci. Food Agric. Samanta, I., S. N. Joardar, P. K. Das, T. K. Sar, S. Bandyopadhyay,
87:2774–2781. T. K. Dutta, and U. Sarkar. 2014. Prevalence and antibiotic re-
Mezal, E. H., A. Sabol, M. A. Khan, N. Ali, R. Stefanova, and sistance profiles of Salmonella serotypes isolated from backyard
A. A. Khan. 2014. Isolation and molecular characterization of poultry flocks in West Bengal, India, India. J. Appl. Poult. Res.
Salmonella enterica serovar Enteritidis from poultry house and 23:536–545.
clinical samples during 2010. Food Microbiol. 38:67–74. Saravanan, S., V. Purushothaman, T. R. G. Murthy, K.
Miao, Z. H., P. C. Glatz, and Y. J. Ru. 2005. Free-range poultry Sukumar, P. Srinivasan, V. Gowthaman, M. Balusamy, R.
production − a review. Asian Australas. J. Anim. Sci 18:113– Atterbury, and S. V. Kuchipudi. 2015. Molecular epidemiology
132. of nontyphoidal Salmonella in poultry and poultry products in
Mikoleit, M. L. 2014. Biochemical identification of Salmonella and India: implications for human health. Indian J. Microbiol. 55:319–
Shigella using an abbreviated panel of tests. World Health 326.
Organization, Global Foodborne Infections Network, Geneva, Sasipreeyajan, J., J. Jerngklinchan, C. Koowatananukul, and K. Sai-
Switzerland. tanu. 1996. Prevalence of Salmonellae in broiler and breeder flocks
Ministry of Health, Malaysia. 2015. Health Facts 2015. Health Infor- in Thailand. Trop. Anim. Health Prod. 28:174–180.
matics Centre, Planning and Development Division, Ministry of Shah, D. H., N. C. Paul, W. C. Sischo, R. Crespo, and J. Guard.
Health, Malaysia, Kuala Lumpur, Malaysia. 2016. Population dynamics and antimicrobial resistance of the
Murakami, K., T. Noda, D. Onozuka, and N. Sera. 2013. Salmonella most prevalent poultry-associated Salmonella serotypes. Poult.
in liquid eggs and other foods in Fukuoka Prefecture, Japan. Int. Sci. 96:687–702.
J. Microbiol. 2013:1–5. Singh, R., A. S. Yadav, V. Tripathi, and R. P. Singh. 2013. An-
Mustaffa, S. S., A. A. Saleha, and A. Jalila. 2014. Occurrence of timicrobial resistance profile of Salmonella present in poultry
antibiotic resistant Salmonella and Campylobacter in wild birds. and poultry environment in north India. Food Control 33:545–
J. Vet. Malaysia 26:17–19. 548.
Najwa, M. S., Y. Rukayadi, A. Ubong, Y. Y. Loo, W. S. Chang, Thung, T. Y., N. A. Mahyudin, D. F. Basri, C. W. J. Wan
Y. L. Lye, T. Y. Thung, S. A. Aimi, T. T. H. Malcolm, S. G. Mohamed Radzi, Y. Nakaguchi, M. Nishibuchi, and S. Radu.
Goh, C. H. Kuan, N. Yoshitsugu, M. Nishibuchi, and R. Son. 2016. Prevalence and antibiotic resistance of Salmonella enteri-
2015. Quantification and antibiotic susceptibility of Salmonella tidis and Salmonella typhimurium in raw chicken meat at retail
spp., Salmonella enteritidis and Salmonella typhimurium in raw markets in Malaysia. Poult. Sci. 95:1888–1893.
vegetables (ulam). Int. Food Res. J. 22:1761–1769. Vanselow, B., M. Hornitzky, K. Walker, G. Eamens, G. Bailey, P.
Nidaullah, H., N. Abirami, A. K. Shamila-Syuhada, L.-O. Chuah, Gill, K. Coates, B. Corney, J. Cronin, and S. Renilson. 2007.
H. Nurul, T. P. Tan, F. W. Zainal Abidin, and G. Rusul. 2017. Salmonella and on-farm risk factors in healthy slaughter-age
Prevalence of Salmonella in poultry processing environments in cattle and sheep in eastern Australia. Aust. Vet. J. 85:498–
wet markets in Penang and Perlis, Malaysia. Vet. World 10:286– 502.
292. Wang, Y., B. Yang, Y. Wu, Z. Zhang, X. Meng, M. Xi, X. Wang,
Okoli, C. I., G. E. Ndujihe, and I. P. Ogbuewu. 2006. Frequency X. Xia, X. Shi, D. Wang, and J. Meng. 2015. Molecular charac-
of isolation of salmonella from commercial poultry feeds and terization of Salmonella enterica serovar Enteritidis on retail raw
their anti-microbial resistance profiles, Imo State, Nigeria. Online poultry in six provinces and two National cities in China. Food
J. Health Allied Sci. 5. http://www.ojhas.org/issue18/2006-2-3. Microbiol. 46:74–80.
htm. Whelan, J., H. Noel, I. Friesema, A. Hofhuis, C. M. de Jager,
Ong, L. P., K. Muniandy, S. P. How, L. S. Yip, and B. K. Lim. 2014. M. Heck, A. Heuvelink, and W. van Pelt. 2010. National out-
Salmonella isolation from poultry farms in Malaysia from 2011 to break of Salmonella Typhimurium (Dutch) phage-type 132 in the
2013. Malaysian J. Vet. Res. 5:1–7. Netherlands, October to December 2009. Euro Surveill
5970 JAJERE ET AL.

15 https://www.eurosurveillance.org/content/10.2807/ese.15.44. 193–213 in Current Topics in Salmonella and Salmonellosis. M.

19705-en. Mares, ed. InTechOpen, Rijeka, Croatia.
Yasin, R. M., M. Jegathesan, and C. C. Tiew. 1997. Salmonella Zhang, G., E. Thau, E. W. Brown, and T. S. Hammack. 2013.
serotypes isolated in Malaysia over the ten-year period 1983-1992. Comparison of a novel strategy for the detection and isolation
Asia Pac. J. Public Health 9:1–5. of Salmonella in shell eggs with the Food and Drug Adminis-
Zamora-Sanabria, R., and A. M. Alvarado. 2017. Preharvest tration Bacteriological Analytical Manual method1. Poult. Sci.
Salmonella risk contamination and the control strategies. Pages 92:3266–3274.