- Email: [email protected]
Salmonella status of table eggs in commercial layer farms in Menoua Division, West region of Cameroon
Accepted Manuscript Salmonella status of table eggs in commercial layer farms in Menoua Division, West region of Cameroon
Marc K. Kouam, Marius.H.F. Biekop, Bridget Katte, Alexis Teguia PII:
S0956-7135(17)30470-X
DOI:
10.1016/j.foodcont.2017.09.037
Reference:
JFCO 5809
To appear in:
Food Control
Received Date:
06 July 2017
Revised Date:
22 September 2017
Accepted Date:
29 September 2017
Please cite this article as: Marc K. Kouam, Marius.H.F. Biekop, Bridget Katte, Alexis Teguia, Salmonella status of table eggs in commercial layer farms in Menoua Division, West region of Cameroon, Food Control (2017), doi: 10.1016/j.foodcont.2017.09.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Highlights
Table eggs are contaminated with Salmonella in commercial layers farms in the West Region of Cameroon.
The Salmonella isolates identified are a threat to consumer’s health and to poultry industry.
The One health approach should be used to ensure the production of safe, Salmonellafree eggs for consumers.
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Salmonella status of table eggs in commercial layer farms in Menoua Division, West region
2
of Cameroon
3 4
Marc K. Kouam*,a,b, Marius.H.F Biekopa, Bridget Kattea, Alexis Teguiaa
5
aDepartment
6
188, Dschang, Cameroon
of Animal Production, Faculty of Agronomy and Agricultural Sciences, P.O. Box
7 8
bCenter
9
Yaoundé, Cameroon
for Research on Filariases and other Tropical Diseases (CRFilMT), P.O. Box
5797,
10 11
________________________________________________
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*Corresponding
author: Marc Kouam, PO Box 188, Dschang; Email: [email protected]
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Abstract
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Egg and poultry industry products are affected by the bacteriological quality of eggs.
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Contamination of edible contents of eggs with Salmonella may either lead to disease outbreaks in
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human or to weak / diseased hatched chicks. In order to obtain a baseline data on the occurrence
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of these bacteria in hen eggs in Cameroon, eggs were collected from layer farms in the West
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Region of Cameroon and examined for Salmonella contamination using standard bacteriological
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methods. A total of 140 table eggs from 20 flocks belonging to 19 layer farms were sampled.
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The mean age of the sampled farms and layers were 11.35±10.30 years and 50.44±23. 07 weeks
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respectively. The flock size varied between 500 and 7,000 layers. All the 19 sampled farms were
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positive for at least one Salmonella isolate. Three serovars were recorded in the flocks: S.
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Enteritidis present in nearly all the sampled flocks (19 out of 20) and on all the farms (19/19), S.
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Paratyphi present in 35% of sampled flocks (7 out of 20), and S. Typhimurium present in 30% of
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sampled flocks (6 out of 20). Over all, the prevalence of Salmonella in the sampled eggs was
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88.6% (124 out of 140). The highest prevalence was obtained with S. Enteritidis (75.7%),
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followed by S. Paratyphi (8.6%) and finally S. Typhimurium (4.3%). S. Enteritidis was found to
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be the most widespread isolate, recorded in all the sampled localities. The occurrence of these
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zoonotic Salmonella serovars constitutes a big challenge for poultry and egg industry, and for
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public health.
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Key words: Salmonella, table eggs, layer, farms, Cameroon.
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1. INTRODUCTION
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Eggs and egg-products are among the most known and consumed food worldwide
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regardless of age, tribe, nationality or religion.. However, one of the constraints limiting the full
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acceptance of egg and egg-products is the suspicion about their bacteriological integrity. Among
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bacteria, Salmonella is one of the mostly isolated contaminating agents in eggs, surely due to its
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ability to be transmitted both vertically (from layer to egg) and horizontally (from environment)
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(Gantois et al., 2009; Gast et al., 2014). Contamination of eggs with Salmonella is both of public
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health and poultry and egg industry concern. Regarding poultry industry, internal contamination
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of eggs with Salmonella in the reproductive organs during egg formation leads in case of fertile
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eggs, to Salmonella contamination of day old chicks. These chicks in turn could serve as an
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extensive bacterial reservoir in commercial hatcheries (Cox et al., 2000) or on farms (Corrier et
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al., 1995). Salmonella in eggs has also been reported to lead to poor egg quality and viability,
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weak day old chicks and high mortality in infected broilers and layers (OIE, 2012). In addition,
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hens infected with Salmonella have been shown to exhibit a decreased egg production (Gantois
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et al., 2008).
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The negative impact of Salmonella on egg industry is the result of the negative publicity
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from the consumer’s perception that eggs are linked to human salmonellosis. It also arises from
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the additional production cost associated with the implementation of Salmonella control program
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or Salmonella treatment on layer farms; in Australia for instance, the negative consumer’s
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confidence in eggs following numerous egg-related human salmonellosis prompted a holding of
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nationwide workshops on Salmonella and eggs issues (Chousalkar et al., 2015). 3
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About the public health concern, Salmonella contamination of eggs or related products
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has been pointed out as one of the most important causes of salmonellosis outbreak in human
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worldwide (Curri et al.,2005; Havelaar et al., 2013).
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In Cameroon, the consumption of eggs has been increasing; in 2012, the total egg
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production reached a quantity of 63,382 tons which was up by 8.8% from the previous year
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(NIS, 2013). In 2009, the egg consumption was 0.95 kg/inhabitant/year, and the demand was
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expected to increase with the growing population (Minepia, 2009). Currently, the prophylaxis
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program enforced on local farms does not consider Salmonella infections, though the egg
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production system (floor housing system) added to poor hygiene on farms is conducive to
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Salmonella occurrence and transmission (De Vylder et al., 2011; Parisi et al., 2015). The current
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outbreak of bird flu affecting many farms in the country suggest that the biosecurity measures
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are poor and that conditions are set for Salmonella spread on farms. In addition, a Salmonella
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control program has never been implemented on poultry farms in the country as in other Nations
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such as Korea. For instance, a vaccine program in Korea using the live S. Gallinarum 9R strain to
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control fowl typhoid caused by S. Gallinarum drastically reduced the outbreaks of fowl typhoid
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in layer chicken (Im et al., 2015). At the same time a cross-protection between S. Gallinarum and
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S. Enteritidis was observed after vaccination (Im et al., 2015), leading to a reduction in the
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occurrence of S. Enteritidis, the zoonotic agent traditionally associated with eggs and egg
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products (Van Hoorebeke, 2011). Though the risk of contamination with Salmonella currently
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does not cause the Cameroon people to have any aversion to eggs locally produced, considering
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the leadership position of Cameroon in terms of table eggs production in Central Africa, and
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taking into account this era of globalization of economy and information, it is important to gain
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knowledge regarding the Salmonella status of eggs locally produced in order to anticipate 4
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appropriately. Therefore, the objective of this study was three-fold: a) to present a pictorial view
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of layers’ farms characteristics in the area, b) to determine the Salmonella status of eggs
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produced in these layer farms, and c) to identify the Salmonella isolates occurring in the eggs.
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2. MATERIAL AND METHOD
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2.1. Study Area
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The study was carried out in Menoua Division of the West Region of Cameroon (Fig. 1)
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from May to July 2016. Animal husbandry in the Division consists of rearing small and large
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ruminants, cavies, pigs, rabbits, broilers and layers among others. The West region is the greatest
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poultry and egg production area in Cameroon, and even in Central Africa, with 53,992 tons of
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eggs out of 63,382 tons in total produced country-wide (NIS, 2013). The housing system adopted
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by farmers is the deep litter system. In this system, hens freely move on a floor covered with
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wood shaving.
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2.2. Questionnaire Design
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The questionnaire was filled in following an on-farm interview at the time of sampling.
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Questions related to farmer (age, experience in animal husbandry), farm (age, duration of
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sanitation between two production cycles) and flock characteristics (flock size, bird age). The
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questionnaire was pre-tested and adjusted accordingly.
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2.3. Sample Collection
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Basic information on the location of farms was provided by the veterinary health service
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of the Subdivision. Then the snow-ball technique was used to identify the farms to be included in
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the study. Once a farm was identified, the owner was contacted and only volunteer farmers
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participated in the study. Due to negative publicity about bird flu epizootic occurring in the
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country during the sampling period, many farmers rejected our request to visit their farms.
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On farm sampling consist of a random collection of 7 eggs from 7 laying nests evenly
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selected throughout the poultry pen. Eggs were collected with gloved hands, introduced in sterile
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plastic bags and transported to the laboratory inside a cool-box.
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2.4. Bacteriological Analysis
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The samples were analyzed using the classical method. Sterile cotton was dipped into 75
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% alcohol and used to swab the entire surface of the egg. Egg surface was air-dried and cracked
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using a sterile knife. Each egg’s content was thoroughly mixed and 1 mL of this mixed content
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was inoculated into 9 mL of buffered peptone water, homogenized and incubated at 37±1 °C for
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24h. Next, 1mL of the pre-enrichment culture was inoculated into selenite cystine broth and
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incubated at 37°±1°C for 24 hours, before plating out onto a selective agar. A drop of the
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selective enrichment culture was plated onto Salmonella-Shigella (SS) agar. Plates were
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incubated in an inverted position for 24h at 37±1°C, and Salmonella-positive plates, based on
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presence of typical colonies, were recorded. Salmonella-like colonies were gram stained,
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inoculated into lactose and glucose broth. The resulting gram negative rods, non lactose
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fermenting but glucose fermenting with gaz production were further characterized under a
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biochemical test using the kligler’s iron agar (KIA), motility indole urea (MIU) and Simmon’s
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citrate test. . The colonies were inoculated into sterile tubes containing 3 mL of these media, 6
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and incubated over night at 37±1°C for 24 hours.. Bacteria serovars formally classified as species
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were identified based on their biochemical reaction and their motility speed (Cheesbrough,
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1984). With the KIA test, S. paratyphi produces gas buble while S. Typhimurium produces gas
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bubble and H2S (Cheesbrough, 1984). S. Enteritidis is H2S producing under KIA test, yields a
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pink color under MIU test and produces no reaction under Simmons’ citrate test. In addition,
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under a microcope, S. Typhimurium is faster than S. Enteritidis while S. Paratyphi is the slowest
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of the three.
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2.5. Statistical Analysis Descriptive statistics were used to summarize the data. A flock or farm was classified as
135 136
infected if the egg sample from the farm was positive for any Salmonella isolate. All statistics
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(means, percentage) were performed using SPSS statistical package version 13.0 (, SPSS Inc.,
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USA).
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3. RESULTS
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A total of 140 table eggs were sampled from 20 flocks belonging to 19 laying hen farms.
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The main characteristics of sampled animals, farms and farmers are presented in Table 1. The
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mean farmer’s age and experience in laying hen farming were 44.89±10.57 and 10.33±6.96 years
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respectively. Similarly, the mean age of the sampled farms and layers were 11.35±10.30 years
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and 50.44±23.07 weeks respectively. The flock size varied between 500 to 7,000 layers, and the
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sanitation period in between two production rounds varied between 3 to 7 months.
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All the 19 sampled farms were positive for at least one Salmonella isolate (Table 2). The within-flock infection rate varied from 28.6 to 100 %. Among localities, the within flock 7
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infection rate varied greatly from 71.4 to 100% in Nkong-ni, 28.6 to 100% in Dschang, and 100
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% in all flocks in Santchou. Three serovars were recorded in the flocks: S. Enteritidis present in
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nearly all the sampled flocks (19 out of 20) and on all the farms (19/19), S. Paratyphi present in
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35% of sampled flocks (7 out of 20), and S. Typhimurium present in 30% of sampled flocks (6
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out of 20).
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Over all, the prevalence of Salmonella in the sampled eggs was 88.6% (124 out of 140).
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The highest prevalence was obtained with S. Enteritidis (75.7%), followed by S. Paratyphi
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(8.6%) and finally S. Typhimurium (4.3%). S. Enteritidis was found to be the most widespread
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serovars, recorded in all the sampled localities, while S. Typhimurium was located only in
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Nkong-ni (Table 3).
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4. DISCUSSION Overall, the main farm, farmer and animal characteristics showed that layers husbandry is
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an old, well established activity which may justify the lead position of Cameroon in central
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Africa in terms of poultry and table egg production. Based on the young age of the newly
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established farms (1 year) in comparison with old farms (40 years), one could deduce that the
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poultry and egg sector in Cameroon is dynamic and profitable, in spite of the lower number of
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hens in the flocks compared with their European counterpart whose flock size vary between
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7,260 to 959,600 hens (Van Hoorebeke et al., 2010). Thus, it was necessary for the sake of egg
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industry in the country to carry out this study on the presence of Salmonella in eggs
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acknowledged as one of the important constraints to egg and poultry industry worldwide (EFSA,
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2007; FDA, 2009).
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Salmonella was found in almost all the flocks and on all farms sampled, showing that the
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infection is endemic in the study area. These results contrast with a previous finding from Korea
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(Im et al., 2015) where 50.7 % of the flock and 59.3% of the farms examined for egg
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contamination with Salmonella were positive. The widespread distribution of Salmonella in this
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study could be explained by a combination of several factors including poor biosecurity practices
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on farms. Poor implementation of biosecurity measures (including the presence of a fence around
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the farm or the clearing of the farm environment from trees, as is the case in most visited farms)
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may increase the likelihood of farm contamination with Salmonella. Indeed wild animals such as
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mice, rats, cats and even insects reported to carry Salmonella (Liebana et al., 2003; Kinde et al.,
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2005; Carrique-Mas et al., 2009) could easily get access to unfenced farms and contaminate the
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farm environment. Taking advantage of trees, wild birds could also contaminate the farm
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surroundings and environment with Salmonella (Noe et al., 2014). The infection rate of eggs within flocks and locality varied in this study; the variation
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might be due to intrinsic factors of each flock such as the health status of the individual layer ,
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and the contamination level of the environment. However, the fact that the within flock infection
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rate in only 2 flocks was below 50% (28.6 and 42%) suggest that the vast majority of layers
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either shed Salmonella or have the reproductive organs contaminated; this also suggest that only
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a limited environmental space is Salmonella free to obtain only two flocks with a low infection
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rate.
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The prevalence of Salmonella in egg samples was very high, with S. Enteritidis having
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the highest prevalence (75.7%), and S. Typhimiurium the lowest (4.3%). The overall prevalence
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(88.6%) was much higher than the prevalence observed by other authors in Korea (5.2%)( Im et
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al., 2015), Nigeria (20.4%)( Ifeanyichukwu et al., 2016), and Ethiopia (10.5%) (Assefa et al.,
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2011). Several reasons might explain such a high prevalence of Salmonella in table eggs. a) The
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deep litter system in place in the sampled farms causes the hens to remain in contact with the
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feces, thus increasing the risk of Salmonella transmission. Even though in this system hens laid
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their eggs in a dedicated site (laying nest), hens may carry the bacteria to the laying nest once
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contaminated; b) inefficient cleaning and disinfection due to the nature of the building materials
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(bamboo, ground-made blocks), wear of production materials (feeders, drinking tank or pipe,
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egg nests and so forth) and building; c) the presence of pests in a layier house increases the level
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of environmental contamination and maintain the infection from one production round to the
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next (Kopanic et al., 1994; Davies and Breslin, 2003a,b); d) the absence of Salmonella
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vaccination leaves the laying hens totally unprotected against Salmonella; indeed the use of
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Salmonella vaccines in laying hens has been shown to reduce the shedding and colonization of
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the reproductive tract, thus leading to a decrease in the number of internally contaminated eggs
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(Gantois et al., 2006).
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In accordance with other studies (Gantois et al., 2006; Delmas, et al., 2006) S. Enteritidis was the
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most common isolate found in eggs in this study. Some difference in the epidemiology of
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isolates may explain the predominance of S. Enteritidis over other isolates. S. Enteritidis is able
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to adhere better to the reproductive tract mucosa than S. Typhimurium (Wales and Davies,
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2011). The design of the laying hen house to promote natural ventilation (the lower part of the
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house is made up of bricks or bamboo while the upper part of the wall is made up of wire netting 10
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to allow free air movement inside the house) probably enhances the airborne transmission of S.
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Enteritidis. In fact, the airborne transmission of S. Enteritidis in poultry houses has been
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described (Gast et al., 1998). S. Paratyphi recorded in this work is rather uncommon in eggs but
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other isolates than S. Enteritidis and S. Typhimurium have also been documented in eggs
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elsewhere (Im et al., 2015). In addition, S. Paratyphi has been documented in human in
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Cameroon, suggesting that human contamination with S. Paratyphi might be due to consumption
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of contaminated eggs or egg products, since it is common practice in the country to consume raw
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eggs through salad dishes or crudités. S. Typhimurium is the most common isolated bacteria in
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human cases of salmonellosis (Chousalkar et al., 2015) in Australia but in other part of the
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world, both S. Enteritidis and S. Typhimurium are the most common serovars involved in
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Salmonella outbreaks in humans (Currie et al., 2005; Havelaar et al., 2013).
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The presence of S .Enteritidis and S. Typhimurium in the study area indicates that the local population is exposed to salmonellosis outbreaks. In conclusion, the presence of zoonotic Salmonella isolates in table eggs constitutes a big
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challenge to egg industry and public health. To avoid economic disaster similar to the one
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induced by the epidemic of bird flu in poultry sector, it is imperative for the regulatory and
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public health authorities in conjunction with egg producers to conduct workshops aiming at
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developing effective control strategies against Salmonella in the poultry and egg sector.
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Conflict of interest: The authors declare that they have no conflict of interest.
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Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Van Hoorebeke, S., Van Immerseel, F., Schulz, J., Hartung, J., Harisberger, M., Barco, L., Ricci,
337
A., Theodoropoulos, G., Xylouri, E., De Vylder, J., Ducatelle, R., Haesebrouck, F.,
338
Pasmans, F., de Kruif., A and Dewulf, J. (2010). Determination of the within and
339
between flock prevalence and identification of risk factors for Salmonella infections in
340
laying hen flocks housed in conventional and alternative systems. Preventive Veterinary
341
Medecine, 94, 94–100.
342 343
Wales, A. and Davies, R. (2011). A critical review of Salmonella Typhimurium infection in laying hens. Avian Pathology, 40, 429–436.
344 345 346 347 348 349 350 351 352 353 354
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356
Table 1. Main socio-economic characteristics of the sampled farms.
Ages of farmer (years) Experience of farmer in poultry husbandry ( years) Age of infrastructure ( years) Age of hens (weeks) Flock size Duration of sanitation in between production rounds (months) 1 SD= Standard deviation
Mean ±SD1 Minimum 44.89±10.57 30 10.33±6.96 1 11.35±10.30 1 50.44±23.07 16 2337.04±1420.80 500 4.54±1.33 3
Maximum 70 30 40 120 7000 7
357 358 359 360 361 362 363 364 365 366 367 368 18
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Table 2. Detailed overview of the layer flocks found positive for Salmonella Locality
N positive/N of samples
Within flock infection rate (95% CI)
Salmonella serovars Enteritidis Paratyphi Typhimurium
370
Nkong-ni
7/7
100% (100-100%)
positive
negative
positive
Nkong-ni
7/7
100% (100-100%)
positive
positive
positive
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Nkong-ni
5/7
71.4% (29-96.3%)
positive
negative
positive
Nkong-ni
6/7
85.7 % (42.1-99.6%)
positive
negative
positive
Nkong-ni
6/7
85.7 % (42.1-99.6%)
positive
positive
positive
Nkong-ni
7/7
100% (100-100%)
positive
negative
positive
Nkong-ni
7/7
100% (100-100%)
positive
positive
negative
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Nkong-ni
7/7
100% (100-100%)
positive
positive
negative
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Dschang
6/7
85.7 % (42.1-99.6%)
positive
negative
negative
Dschang
7/7
100% (100-100%)
positive
negative
negative
Dschang
6/7
85.7 % (42.1-99.6%)
positive
negative
negative
Dschang
3/7
42 (9.9-81.6 )
negative
positive
negative
Dschang
2/7
28.6% (3.7-71%)
positive
positive
negative
Dschang
6/7
85.7 % (42.1-99.6%)
positive
positive
negative
Santchou
7/7
100% (100-100%)
positive
negative
negative
Santchou
7/7
100% (100-100%)
positive
negative
negative
CI: confidence interval 19
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371 372
Table 3. Prevalence (%) of Salmonella per locality in Menoua Division Salmonella isolate Enteritidis Paratyphi Typhimurium Total N n (%) n (%) n (%) Dshang 42 23 (54.8) 7(16.7) 0 (0.0) 30(71.4) Nkong-ni 84 69 (82.1) 5(6.0) 6(7.1) 80(95.2) Santchou 14 14 (100) 0 (0.0) 0 (0.0) 14(100.0) Total 140 106 (75.7) 12 (8.6) 6 (4.3) 124 (88.6) N= Total number of sample; n= number of sample positive for Salmonella Locality
373 374 375 376 377 378 379
Figure caption
380 381
Figure 1. Map of the West region of Cameroon showing Menoua Division.
382 383
20
Marc K. Kouam, Marius.H.F. Biekop, Bridget Katte, Alexis Teguia PII:
S0956-7135(17)30470-X
DOI:
10.1016/j.foodcont.2017.09.037
Reference:
JFCO 5809
To appear in:
Food Control
Received Date:
06 July 2017
Revised Date:
22 September 2017
Accepted Date:
29 September 2017
Please cite this article as: Marc K. Kouam, Marius.H.F. Biekop, Bridget Katte, Alexis Teguia, Salmonella status of table eggs in commercial layer farms in Menoua Division, West region of Cameroon, Food Control (2017), doi: 10.1016/j.foodcont.2017.09.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
Table eggs are contaminated with Salmonella in commercial layers farms in the West Region of Cameroon.
The Salmonella isolates identified are a threat to consumer’s health and to poultry industry.
The One health approach should be used to ensure the production of safe, Salmonellafree eggs for consumers.
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1
Salmonella status of table eggs in commercial layer farms in Menoua Division, West region
2
of Cameroon
3 4
Marc K. Kouam*,a,b, Marius.H.F Biekopa, Bridget Kattea, Alexis Teguiaa
5
aDepartment
6
188, Dschang, Cameroon
of Animal Production, Faculty of Agronomy and Agricultural Sciences, P.O. Box
7 8
bCenter
9
Yaoundé, Cameroon
for Research on Filariases and other Tropical Diseases (CRFilMT), P.O. Box
5797,
10 11
________________________________________________
12
*Corresponding
author: Marc Kouam, PO Box 188, Dschang; Email: [email protected]
13 14 15 16 17 18 19 1
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Abstract
21
Egg and poultry industry products are affected by the bacteriological quality of eggs.
22
Contamination of edible contents of eggs with Salmonella may either lead to disease outbreaks in
23
human or to weak / diseased hatched chicks. In order to obtain a baseline data on the occurrence
24
of these bacteria in hen eggs in Cameroon, eggs were collected from layer farms in the West
25
Region of Cameroon and examined for Salmonella contamination using standard bacteriological
26
methods. A total of 140 table eggs from 20 flocks belonging to 19 layer farms were sampled.
27
The mean age of the sampled farms and layers were 11.35±10.30 years and 50.44±23. 07 weeks
28
respectively. The flock size varied between 500 and 7,000 layers. All the 19 sampled farms were
29
positive for at least one Salmonella isolate. Three serovars were recorded in the flocks: S.
30
Enteritidis present in nearly all the sampled flocks (19 out of 20) and on all the farms (19/19), S.
31
Paratyphi present in 35% of sampled flocks (7 out of 20), and S. Typhimurium present in 30% of
32
sampled flocks (6 out of 20). Over all, the prevalence of Salmonella in the sampled eggs was
33
88.6% (124 out of 140). The highest prevalence was obtained with S. Enteritidis (75.7%),
34
followed by S. Paratyphi (8.6%) and finally S. Typhimurium (4.3%). S. Enteritidis was found to
35
be the most widespread isolate, recorded in all the sampled localities. The occurrence of these
36
zoonotic Salmonella serovars constitutes a big challenge for poultry and egg industry, and for
37
public health.
38
Key words: Salmonella, table eggs, layer, farms, Cameroon.
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1. INTRODUCTION
42 43
Eggs and egg-products are among the most known and consumed food worldwide
44
regardless of age, tribe, nationality or religion.. However, one of the constraints limiting the full
45
acceptance of egg and egg-products is the suspicion about their bacteriological integrity. Among
46
bacteria, Salmonella is one of the mostly isolated contaminating agents in eggs, surely due to its
47
ability to be transmitted both vertically (from layer to egg) and horizontally (from environment)
48
(Gantois et al., 2009; Gast et al., 2014). Contamination of eggs with Salmonella is both of public
49
health and poultry and egg industry concern. Regarding poultry industry, internal contamination
50
of eggs with Salmonella in the reproductive organs during egg formation leads in case of fertile
51
eggs, to Salmonella contamination of day old chicks. These chicks in turn could serve as an
52
extensive bacterial reservoir in commercial hatcheries (Cox et al., 2000) or on farms (Corrier et
53
al., 1995). Salmonella in eggs has also been reported to lead to poor egg quality and viability,
54
weak day old chicks and high mortality in infected broilers and layers (OIE, 2012). In addition,
55
hens infected with Salmonella have been shown to exhibit a decreased egg production (Gantois
56
et al., 2008).
57
The negative impact of Salmonella on egg industry is the result of the negative publicity
58
from the consumer’s perception that eggs are linked to human salmonellosis. It also arises from
59
the additional production cost associated with the implementation of Salmonella control program
60
or Salmonella treatment on layer farms; in Australia for instance, the negative consumer’s
61
confidence in eggs following numerous egg-related human salmonellosis prompted a holding of
62
nationwide workshops on Salmonella and eggs issues (Chousalkar et al., 2015). 3
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About the public health concern, Salmonella contamination of eggs or related products
64
has been pointed out as one of the most important causes of salmonellosis outbreak in human
65
worldwide (Curri et al.,2005; Havelaar et al., 2013).
66
In Cameroon, the consumption of eggs has been increasing; in 2012, the total egg
67
production reached a quantity of 63,382 tons which was up by 8.8% from the previous year
68
(NIS, 2013). In 2009, the egg consumption was 0.95 kg/inhabitant/year, and the demand was
69
expected to increase with the growing population (Minepia, 2009). Currently, the prophylaxis
70
program enforced on local farms does not consider Salmonella infections, though the egg
71
production system (floor housing system) added to poor hygiene on farms is conducive to
72
Salmonella occurrence and transmission (De Vylder et al., 2011; Parisi et al., 2015). The current
73
outbreak of bird flu affecting many farms in the country suggest that the biosecurity measures
74
are poor and that conditions are set for Salmonella spread on farms. In addition, a Salmonella
75
control program has never been implemented on poultry farms in the country as in other Nations
76
such as Korea. For instance, a vaccine program in Korea using the live S. Gallinarum 9R strain to
77
control fowl typhoid caused by S. Gallinarum drastically reduced the outbreaks of fowl typhoid
78
in layer chicken (Im et al., 2015). At the same time a cross-protection between S. Gallinarum and
79
S. Enteritidis was observed after vaccination (Im et al., 2015), leading to a reduction in the
80
occurrence of S. Enteritidis, the zoonotic agent traditionally associated with eggs and egg
81
products (Van Hoorebeke, 2011). Though the risk of contamination with Salmonella currently
82
does not cause the Cameroon people to have any aversion to eggs locally produced, considering
83
the leadership position of Cameroon in terms of table eggs production in Central Africa, and
84
taking into account this era of globalization of economy and information, it is important to gain
85
knowledge regarding the Salmonella status of eggs locally produced in order to anticipate 4
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appropriately. Therefore, the objective of this study was three-fold: a) to present a pictorial view
87
of layers’ farms characteristics in the area, b) to determine the Salmonella status of eggs
88
produced in these layer farms, and c) to identify the Salmonella isolates occurring in the eggs.
89 90
2. MATERIAL AND METHOD
91
2.1. Study Area
92
The study was carried out in Menoua Division of the West Region of Cameroon (Fig. 1)
93
from May to July 2016. Animal husbandry in the Division consists of rearing small and large
94
ruminants, cavies, pigs, rabbits, broilers and layers among others. The West region is the greatest
95
poultry and egg production area in Cameroon, and even in Central Africa, with 53,992 tons of
96
eggs out of 63,382 tons in total produced country-wide (NIS, 2013). The housing system adopted
97
by farmers is the deep litter system. In this system, hens freely move on a floor covered with
98
wood shaving.
99
2.2. Questionnaire Design
100
The questionnaire was filled in following an on-farm interview at the time of sampling.
101
Questions related to farmer (age, experience in animal husbandry), farm (age, duration of
102
sanitation between two production cycles) and flock characteristics (flock size, bird age). The
103
questionnaire was pre-tested and adjusted accordingly.
104
2.3. Sample Collection
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Basic information on the location of farms was provided by the veterinary health service
106
of the Subdivision. Then the snow-ball technique was used to identify the farms to be included in
107
the study. Once a farm was identified, the owner was contacted and only volunteer farmers
108
participated in the study. Due to negative publicity about bird flu epizootic occurring in the
109
country during the sampling period, many farmers rejected our request to visit their farms.
110
On farm sampling consist of a random collection of 7 eggs from 7 laying nests evenly
111
selected throughout the poultry pen. Eggs were collected with gloved hands, introduced in sterile
112
plastic bags and transported to the laboratory inside a cool-box.
113
2.4. Bacteriological Analysis
114
The samples were analyzed using the classical method. Sterile cotton was dipped into 75
115
% alcohol and used to swab the entire surface of the egg. Egg surface was air-dried and cracked
116
using a sterile knife. Each egg’s content was thoroughly mixed and 1 mL of this mixed content
117
was inoculated into 9 mL of buffered peptone water, homogenized and incubated at 37±1 °C for
118
24h. Next, 1mL of the pre-enrichment culture was inoculated into selenite cystine broth and
119
incubated at 37°±1°C for 24 hours, before plating out onto a selective agar. A drop of the
120
selective enrichment culture was plated onto Salmonella-Shigella (SS) agar. Plates were
121
incubated in an inverted position for 24h at 37±1°C, and Salmonella-positive plates, based on
122
presence of typical colonies, were recorded. Salmonella-like colonies were gram stained,
123
inoculated into lactose and glucose broth. The resulting gram negative rods, non lactose
124
fermenting but glucose fermenting with gaz production were further characterized under a
125
biochemical test using the kligler’s iron agar (KIA), motility indole urea (MIU) and Simmon’s
126
citrate test. . The colonies were inoculated into sterile tubes containing 3 mL of these media, 6
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and incubated over night at 37±1°C for 24 hours.. Bacteria serovars formally classified as species
128
were identified based on their biochemical reaction and their motility speed (Cheesbrough,
129
1984). With the KIA test, S. paratyphi produces gas buble while S. Typhimurium produces gas
130
bubble and H2S (Cheesbrough, 1984). S. Enteritidis is H2S producing under KIA test, yields a
131
pink color under MIU test and produces no reaction under Simmons’ citrate test. In addition,
132
under a microcope, S. Typhimurium is faster than S. Enteritidis while S. Paratyphi is the slowest
133
of the three.
134
2.5. Statistical Analysis Descriptive statistics were used to summarize the data. A flock or farm was classified as
135 136
infected if the egg sample from the farm was positive for any Salmonella isolate. All statistics
137
(means, percentage) were performed using SPSS statistical package version 13.0 (, SPSS Inc.,
138
USA).
139
3. RESULTS
140
A total of 140 table eggs were sampled from 20 flocks belonging to 19 laying hen farms.
141
The main characteristics of sampled animals, farms and farmers are presented in Table 1. The
142
mean farmer’s age and experience in laying hen farming were 44.89±10.57 and 10.33±6.96 years
143
respectively. Similarly, the mean age of the sampled farms and layers were 11.35±10.30 years
144
and 50.44±23.07 weeks respectively. The flock size varied between 500 to 7,000 layers, and the
145
sanitation period in between two production rounds varied between 3 to 7 months.
146 147
All the 19 sampled farms were positive for at least one Salmonella isolate (Table 2). The within-flock infection rate varied from 28.6 to 100 %. Among localities, the within flock 7
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infection rate varied greatly from 71.4 to 100% in Nkong-ni, 28.6 to 100% in Dschang, and 100
149
% in all flocks in Santchou. Three serovars were recorded in the flocks: S. Enteritidis present in
150
nearly all the sampled flocks (19 out of 20) and on all the farms (19/19), S. Paratyphi present in
151
35% of sampled flocks (7 out of 20), and S. Typhimurium present in 30% of sampled flocks (6
152
out of 20).
153
Over all, the prevalence of Salmonella in the sampled eggs was 88.6% (124 out of 140).
154
The highest prevalence was obtained with S. Enteritidis (75.7%), followed by S. Paratyphi
155
(8.6%) and finally S. Typhimurium (4.3%). S. Enteritidis was found to be the most widespread
156
serovars, recorded in all the sampled localities, while S. Typhimurium was located only in
157
Nkong-ni (Table 3).
158 159 160
4. DISCUSSION Overall, the main farm, farmer and animal characteristics showed that layers husbandry is
161
an old, well established activity which may justify the lead position of Cameroon in central
162
Africa in terms of poultry and table egg production. Based on the young age of the newly
163
established farms (1 year) in comparison with old farms (40 years), one could deduce that the
164
poultry and egg sector in Cameroon is dynamic and profitable, in spite of the lower number of
165
hens in the flocks compared with their European counterpart whose flock size vary between
166
7,260 to 959,600 hens (Van Hoorebeke et al., 2010). Thus, it was necessary for the sake of egg
167
industry in the country to carry out this study on the presence of Salmonella in eggs
8
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acknowledged as one of the important constraints to egg and poultry industry worldwide (EFSA,
169
2007; FDA, 2009).
170
Salmonella was found in almost all the flocks and on all farms sampled, showing that the
171
infection is endemic in the study area. These results contrast with a previous finding from Korea
172
(Im et al., 2015) where 50.7 % of the flock and 59.3% of the farms examined for egg
173
contamination with Salmonella were positive. The widespread distribution of Salmonella in this
174
study could be explained by a combination of several factors including poor biosecurity practices
175
on farms. Poor implementation of biosecurity measures (including the presence of a fence around
176
the farm or the clearing of the farm environment from trees, as is the case in most visited farms)
177
may increase the likelihood of farm contamination with Salmonella. Indeed wild animals such as
178
mice, rats, cats and even insects reported to carry Salmonella (Liebana et al., 2003; Kinde et al.,
179
2005; Carrique-Mas et al., 2009) could easily get access to unfenced farms and contaminate the
180
farm environment. Taking advantage of trees, wild birds could also contaminate the farm
181
surroundings and environment with Salmonella (Noe et al., 2014). The infection rate of eggs within flocks and locality varied in this study; the variation
182 183
might be due to intrinsic factors of each flock such as the health status of the individual layer ,
184
and the contamination level of the environment. However, the fact that the within flock infection
185
rate in only 2 flocks was below 50% (28.6 and 42%) suggest that the vast majority of layers
186
either shed Salmonella or have the reproductive organs contaminated; this also suggest that only
187
a limited environmental space is Salmonella free to obtain only two flocks with a low infection
188
rate.
9
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189
The prevalence of Salmonella in egg samples was very high, with S. Enteritidis having
190
the highest prevalence (75.7%), and S. Typhimiurium the lowest (4.3%). The overall prevalence
191
(88.6%) was much higher than the prevalence observed by other authors in Korea (5.2%)( Im et
192
al., 2015), Nigeria (20.4%)( Ifeanyichukwu et al., 2016), and Ethiopia (10.5%) (Assefa et al.,
193
2011). Several reasons might explain such a high prevalence of Salmonella in table eggs. a) The
194
deep litter system in place in the sampled farms causes the hens to remain in contact with the
195
feces, thus increasing the risk of Salmonella transmission. Even though in this system hens laid
196
their eggs in a dedicated site (laying nest), hens may carry the bacteria to the laying nest once
197
contaminated; b) inefficient cleaning and disinfection due to the nature of the building materials
198
(bamboo, ground-made blocks), wear of production materials (feeders, drinking tank or pipe,
199
egg nests and so forth) and building; c) the presence of pests in a layier house increases the level
200
of environmental contamination and maintain the infection from one production round to the
201
next (Kopanic et al., 1994; Davies and Breslin, 2003a,b); d) the absence of Salmonella
202
vaccination leaves the laying hens totally unprotected against Salmonella; indeed the use of
203
Salmonella vaccines in laying hens has been shown to reduce the shedding and colonization of
204
the reproductive tract, thus leading to a decrease in the number of internally contaminated eggs
205
(Gantois et al., 2006).
206
In accordance with other studies (Gantois et al., 2006; Delmas, et al., 2006) S. Enteritidis was the
207
most common isolate found in eggs in this study. Some difference in the epidemiology of
208
isolates may explain the predominance of S. Enteritidis over other isolates. S. Enteritidis is able
209
to adhere better to the reproductive tract mucosa than S. Typhimurium (Wales and Davies,
210
2011). The design of the laying hen house to promote natural ventilation (the lower part of the
211
house is made up of bricks or bamboo while the upper part of the wall is made up of wire netting 10
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to allow free air movement inside the house) probably enhances the airborne transmission of S.
213
Enteritidis. In fact, the airborne transmission of S. Enteritidis in poultry houses has been
214
described (Gast et al., 1998). S. Paratyphi recorded in this work is rather uncommon in eggs but
215
other isolates than S. Enteritidis and S. Typhimurium have also been documented in eggs
216
elsewhere (Im et al., 2015). In addition, S. Paratyphi has been documented in human in
217
Cameroon, suggesting that human contamination with S. Paratyphi might be due to consumption
218
of contaminated eggs or egg products, since it is common practice in the country to consume raw
219
eggs through salad dishes or crudités. S. Typhimurium is the most common isolated bacteria in
220
human cases of salmonellosis (Chousalkar et al., 2015) in Australia but in other part of the
221
world, both S. Enteritidis and S. Typhimurium are the most common serovars involved in
222
Salmonella outbreaks in humans (Currie et al., 2005; Havelaar et al., 2013).
223 224 225
The presence of S .Enteritidis and S. Typhimurium in the study area indicates that the local population is exposed to salmonellosis outbreaks. In conclusion, the presence of zoonotic Salmonella isolates in table eggs constitutes a big
226
challenge to egg industry and public health. To avoid economic disaster similar to the one
227
induced by the epidemic of bird flu in poultry sector, it is imperative for the regulatory and
228
public health authorities in conjunction with egg producers to conduct workshops aiming at
229
developing effective control strategies against Salmonella in the poultry and egg sector.
230 231
Conflict of interest: The authors declare that they have no conflict of interest.
11
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Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
234
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Table 1. Main socio-economic characteristics of the sampled farms.
Ages of farmer (years) Experience of farmer in poultry husbandry ( years) Age of infrastructure ( years) Age of hens (weeks) Flock size Duration of sanitation in between production rounds (months) 1 SD= Standard deviation
Mean ±SD1 Minimum 44.89±10.57 30 10.33±6.96 1 11.35±10.30 1 50.44±23.07 16 2337.04±1420.80 500 4.54±1.33 3
Maximum 70 30 40 120 7000 7
357 358 359 360 361 362 363 364 365 366 367 368 18
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Table 2. Detailed overview of the layer flocks found positive for Salmonella Locality
N positive/N of samples
Within flock infection rate (95% CI)
Salmonella serovars Enteritidis Paratyphi Typhimurium
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Nkong-ni
7/7
100% (100-100%)
positive
negative
positive
Nkong-ni
7/7
100% (100-100%)
positive
positive
positive
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Nkong-ni
5/7
71.4% (29-96.3%)
positive
negative
positive
Nkong-ni
6/7
85.7 % (42.1-99.6%)
positive
negative
positive
Nkong-ni
6/7
85.7 % (42.1-99.6%)
positive
positive
positive
Nkong-ni
7/7
100% (100-100%)
positive
negative
positive
Nkong-ni
7/7
100% (100-100%)
positive
positive
negative
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Nkong-ni
7/7
100% (100-100%)
positive
positive
negative
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Nkong-ni
7/7
100% (100-100%)
positive
negative
negative
Dschang
6/7
85.7 % (42.1-99.6%)
positive
negative
negative
Dschang
7/7
100% (100-100%)
positive
negative
negative
Dschang
6/7
85.7 % (42.1-99.6%)
positive
negative
negative
Dschang
3/7
42 (9.9-81.6 )
negative
positive
negative
Dschang
2/7
28.6% (3.7-71%)
positive
positive
negative
Dschang
6/7
85.7 % (42.1-99.6%)
positive
positive
negative
Santchou
7/7
100% (100-100%)
positive
negative
negative
Santchou
7/7
100% (100-100%)
positive
negative
negative
CI: confidence interval 19
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Table 3. Prevalence (%) of Salmonella per locality in Menoua Division Salmonella isolate Enteritidis Paratyphi Typhimurium Total N n (%) n (%) n (%) Dshang 42 23 (54.8) 7(16.7) 0 (0.0) 30(71.4) Nkong-ni 84 69 (82.1) 5(6.0) 6(7.1) 80(95.2) Santchou 14 14 (100) 0 (0.0) 0 (0.0) 14(100.0) Total 140 106 (75.7) 12 (8.6) 6 (4.3) 124 (88.6) N= Total number of sample; n= number of sample positive for Salmonella Locality
373 374 375 376 377 378 379
Figure caption
380 381
Figure 1. Map of the West region of Cameroon showing Menoua Division.
382 383
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