F1000ResearchF1000Research2046-1402F1000 Research LimitedLondon, UK10.12688/f1000research.18233.1Research ArticleArticlesPrevalence and diversity of
Salmonella isolated from layer farms in central Ecuador
[version 1; peer review: 2 approved with reservations]
SalazarGabriela A.Formal AnalysisInvestigationWriting – Original Draft Preparation1Guerrero-LópezRicardoConceptualizationSupervisionValidationWriting – Original Draft Preparation1LalaleoLilianaFormal AnalysisInvestigationWriting – Review & Editing1Avilés-EsquivelDianaMethodologyValidationWriting – Original Draft Preparationhttps://orcid.org/0000-0003-3819-92061Vinueza-BurgosChristianFormal AnalysisMethodologyWriting – Original Draft Preparationhttps://orcid.org/0000-0002-4893-502X2Calero-CáceresWilliamConceptualizationFormal AnalysisFunding AcquisitionInvestigationMethodologySupervisionWriting – Original Draft PreparationWriting – Review & Editinghttps://orcid.org/0000-0003-3819-9206a1
UTA RAM OneHealth Group, Faculty of Agricultural Sciences, Universidad Técnica de Ambato, Cevallos, Ecuador
Facultad de Medicina Veterinaria y Zootecnia, Universidad Central del Ecuador, Quito, Ecuadorwr.calero@uta.edu.ec
Background: Given the considerable role played by
Salmonella in the incidence of food poisoning around the world, surveillance of this infection is prioritized by both food producers and health care authorities. Data remains insufficient concerning the prevalence of
Salmonella in poultry systems in Ecuador and in Latin America in general.
Methods: In this study we evaluated the prevalence and diversity of
Salmonella serovars in samples taken from 21 layer farms and backyard layers in central Ecuador during August–November 2017.
Salmonella was isolated following standardized methods (ISO 6579) and the serovar determination was carried out by PCR.
Results: A significant presence of
Salmonella was detected, with an incidence of 76% (95% confidence interval (CI): 58–94) in farms, 33% (95%CI: 13–53) in pooled cloacal swabs from layer hens, 33% (95%CI: 12–55) on feed samples, and 10% (95%CI: 0–22) in backyard layer feces from traditional local markets. The dominant serovars detected were
S. Infantis and
S. Typhimurium.
Conclusions: This study forms a basis for further surveillance of
Salmonella serovars in layer farms in central Ecuador.
SalmonellaLayer PoultryEcuadorSerovars.Universidad Técnica de Ambato1568-CU-P-2017This study was supported by the Dirección de Investigación y Desarrollo DIDE-Universidad Técnica de Ambato (Project 1568-CU-P-2017).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Introduction
The genus
Salmonella is considered a leading cause of foodborne illnesses around the world (
WHO, 2017). These bacteria are among the most significant agents of food and water poisoning in the United States and Europe (
Bäumler
et al., 2000;
Callejón
et al., 2015;
Varma
et al., 2005). Globally, it is estimated that 93.8 million cases of
Salmonella-related gastroenteritis occur annually, resulting in 155,000 deaths (
Majowicz
et al., 2010). In Ecuador, typhoid, together with paratyphoid fever, causes around 1,500 hospitalizations per year, while non-typhoidal salmonellosis leads to more than 2,000 hospitalizations over the same period. These infections have accounted for approximately 25% of total reported gastrointestinal illnesses in recent years (
MSP, 2018).
Salmonella represents a complex and diverse genus, but only a small number of serovars are involved in human infections (
Issenhuth-Jeanjean
et al., 2014;
Thiennimitr
et al., 2012).
Salmonella detection and the investigation of foodborne outbreaks is of maximum importance to public health, which has resulted in the establishment of epidemiological surveillance programs in many developed countries (
EFSA, 2013). These measures provide information about endemic
Salmonella-serovar patterns, outbreaks, temporal trends and the monitoring of control actions (
CDC, 2018). The principal reservoir of
S. enterica is the intestinal tract of livestock, representing one of the main sources of infection for humans (
Antunes
et al., 2016). Despite this epidemiological and economic importance, in South America there is a paucity of information concerning
Salmonella in poultry systems (
Alexandre
et al., 2000;
Donado-Godoy
et al., 2012). Research conducted in 2016 pertaining to broiler chicken farming in Ecuador indicated the presence of
Salmonella serotypes
S. Infantis
, S. Enteritidis
and S. Corvallis (
Vinueza-Burgos
et al., 2016). However, among layer hens, no information about the prevalence or diversity of
Salmonella has been reported. The purpose of this study was to assess the prevalence and diversity of
Salmonella bacteria present in layer farms in central Ecuador.
MethodsSamples
Samples were collected between August–November 2017 from different layer farms in central Ecuador (Tungurahua and Cotopaxi provinces), which accounts for around 60% of egg production in the country. A total of 21 farms (>1,000 birds) in Latacunga, Cevallos, Quero and Ambato (all Ecuador) were sampled, based on their willingness to provide verbal consent for this study (verbal consent was obtained over written consent owing to the farmers’ reluctance to sign their names, as they perceived this could be used to identify them). Further details for each site can be found in
Dataset 1 (
Calero-Cáceres, 2019a)). One laying hen house per farm was selected. The following samples were collected in each house: 21 pooled cloacal swabs (10 cloacal swabs per pool); 21 manure drag swabs (environmental swabs, 1 per business); 21 caecum content samples (1 layer per farm). To evaluate the potential risk from contaminated feed, 18 composite samples were taken from farmyards (18 of the 21 farms consented verbally to having these samples taken). Additionally, 21 fecal samples from backyard layers were sampled in traditional local markets. All samples were transported in an icebox at 3–5°C within 2 hours of collection for bacterial isolation. The experiment was performed under supervision of the ethical committee of the Faculty of Agricultural Sciences, Universidad Técnica de Ambato.
Detection of
<italic toggle="yes">Salmonella</italic>
Salmonella was isolated following standardized methods (ISO 6579) (
ISO, 2017). The samples were pre-enriched in buffered peptone water (Oxoid, Basingstoke, England) and then incubated at 37±1°C for 18 h ± 2 h. Next, Rappaport Vassiliadis Soy Broth (RVS Broth) (Merck Millipore, Darmstadt, Germany) was used a selective medium, being inoculated with the pre-enriched culture and incubated at 41.5±1°C for 24±3 h. One loopful of the selective enrichment medium was streaked onto xylose lysine deoxycholate agar (XLD agar) (Becton Dickinson GmbH, Heidelberg, Germany) and incubated at 37±1°C for 24±3 h. Presumptive
Salmonella isolates (identified as red/yellow colonies with a black center) were purified in Mac Conkey agar (Merck, Darmstadt, Germany) and incubated at 37±1°C for 24 h. Isolates were Gram stained and the following biochemical tests were performed for confirmation the genus
Salmonella: a catalase test using 30% hydrogen peroxide (Merck Millipore, Darmstadt, Germany); triple sugar iron agar test (TSI) (Becton Dickinson GmbH, Heidelberg, Germany), Simmons citrate agar test (Merck, Darmstadt, Germany), Christensen urea agar test (Britania Lab., Buenos Aires, Argentina), and indole reaction using tryptone water (Merck, Darmstadt, Germany) and Kovac’s reagent (Sigma Aldrich, St. Louis, USA). One isolate per positive sample was selected and cryopreserved using overnight growth in LB broth (Sigma Aldrich, St. Louis, USA) supplemented with 30% glycerol (Merck Millipore, Darmstadt, Germany) and maintained at -80°C until analysis.
Serovar determination by PCR
PCR assays were performed to identify the genes under specific conditions (
Table 2). DNA was extracted from overnight cultures in Casein-Peptone Soymeal-Peptone Broth (Merck, Darmstadt, Germany) as described by
Muniesa
et al. (2004). Approximately ≈50 ng/reaction resulting from the thermal shock of bacterial suspensions (>10
7 UFC/ml) diluted in sterile ddH
20 was used as template for the PCR reactions. The following reference
Salmonella strains were used as positive controls for the six serovars under investigation:
S. Enteritidis UNIETAR 1,
S. Gallinarum b. Gallinarum NCTC 13346,
S. Gallinarum b. Pullorum ATCC 19945®,
S. Typhi ATCC® 19430,
S. Typhimurium
ATCC® 26930 and
S. Infantis UNIETAR 3CT7.
<italic toggle="yes">Salmonella</italic>-positive samples in relation to evaluated matrices.
Location
Sample
Matrix
Samples, n
Positive samples, n (%)
Farms
Feed
Composite feed
18
6 (33%)
Environment
Environmental swabs
21
16 (76%)
Animal
Laying hen cloacal swab (composite)
21
2 (14%)
Local markets
Environment
Backyard poulty feces
21
7 (33%)
Oligonucleotides used in this study.
Organism
Name and
direction
5’ to 3’ sequence
Gene
Annealing
temperature (°C)
Amplicon
size (bp)
References
Enteritidis
sdf-F
TGT GTT TTA TCT GAT GCA AGA G
sdf locus
56
293
(
Agron
et al., 2001)
sdf-R
CGT TCT TCT GGT ACT TCA GAT GAC
Enterica
bcfC-F
GGG TGG GCG GAA AAC TAT TTC
bcfC
56
993
(
Zhu
et al., 2015)
bcfC-R
CGG CAC GGC GGA ATA GAG CAC
Infantis
M. SinI F
CAC AAT GAA CGT GGT GAA GG
M.Sin I
56
184
(
Ranjbar
et al., 2017)
M. SinI R
TGA ACT ACG TTC GTT CTT CTGG
Gallinarum
biotype
Gallinarum
steB-F
TGT CGA CTG GGA CCC GCC CGC CCG C
steB
56
636
(
Pugliese
et al., 2011)
steB-R
CCA TCT TGT AGC GCA CCA T
Gallinarum
biotype
Pullorum
rhs-F
TCG TTT ACG GCA TTA CAC AAG TA
rhs locus
56
402
(
Zhu
et al., 2015)
rhs-R
CAA ACC CAG AGC CAA TCT TAT CT
Typhi
sty-1
TGC CGG AAA CGA ATC T
23S rRNA gene
53
300
(
Zhu
et al., 1996)
sty-2
GGT TGT CAT GCC AAT GCA CT
Typhimurium
Fli15
CGG TGT TGC CCA GGT TGG TAA T
fliC gene
53
620
(
Pui
et al., 2011)
Typ04
ACT GGT AAA GAT GGC T
The reaction mixture contained: 12.5 µl of DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Massachusetts, USA), 0.5 µl of each primer (30 µM), 9 µl of nuclease-free water (Thermo Fisher Scientific, Massachusetts, USA), and 2.5 µl of crude DNA were used. PCRs were performed with an Applied Biosystems SimplyAmp Thermal Cycler (Thermo Fisher Scientific, Massachusetts, USA). A total of 10 µl of each PCR product were analyzed by agarose gel electrophoresis and stained using Sybr® Safe DNA Gel Stain (Invitrogen, Carlsbad, USA).
ResultsAssessing the presence of
<italic toggle="yes">Salmonella</italic>
Overall, 31 out of 34 isolates which showed phenotypic characteristics in accordance with
Salmonella were confirmed by PCR as
S. enterica (
bcfC gene amplification) (
Table 1). Of the 21 farms, 16 (76%, 95% confidence interval (CI): 58–94) showed the presence of
Salmonella on environmental surfaces, and
Salmonella bacteria were isolated in 7 pooled cloacal swabs (33%, 95%CI: 13–53) and in 6 of 18 composite feed samples (33%, 95%CI: 12–55). Feces from backyard layers showed the presence of
Salmonella in 2 of 21 samples (10%, 95%CI: 0–22).
Dataset 1 shows the location of each sample and the presence or absence of
Salmonella (
Calero-Cáceres, 2019a)
Determining presence of serovars
Regarding to the presence of individual serovars (
Figure 1), the highest occurrence was of
S. Infantis, detected in 58% (18/31) of isolates, followed by
S. Typhimurium, present in 32% of samples (10/31) (
Supplementary Figure 1 (
Calero-Cáceres, 2019c)). The serovar of 10% of
Salmonella isolates (3/31) were unable to be serotyped by the panel of tests. In poultry feed samples,
S. Infantis was present in 100% (6/6), while in
S. enterica isolated from manure drag swabs,
S. Infantis was detected in 50% (8/8) and
S. Typhimurium in 50% (8/8). In backyard poultry feces from local markets,
S. Infantis was detected in 100% of positive samples (2/2). The most heterogeneous diversity of
Salmonella serotypes was observed in pooled cloacal swabs, with 2
S. Infantis, 2 S. Typhimurium and 3
S. enterica consisting of serovars not covered within the panel.
Dataset 1 lists the identity of the serovar taken from each sampling location (
Calero-Cáceres, 2019a).
Dataset 2 shows PCR gels for confirmation of
Salmonella serovars (
Calero-Cáceres, 2019b).
Distribution of
<italic toggle="yes">Salmonella</italic> serovars according to the evaluated matrices.
Discussion
The overall results showed a significant presence of
Salmonella in layer farms in the central Ecuador region, one of the main sources of egg production in the country, with an incidence of more than 76% of the evaluated farms. This result is similar to that reported in Colombia (65%) (
Donado-Godoy
et al., 2012), and considerably higher than the presence of
Salmonella in broiler chicken farms in northern Ecuador (15.9%) (
Vinueza-Burgos
et al., 2016).
The notable presence of
Salmonella in poultry feed suggests that this may constitute a potential reservoir of this bacteria for poultry systems in the evaluated region, and consequently, a potential route of infection and colonization of poultry and subsequent entry into the food chain (
Fink-Gremmels, 2012;
Jones, 2011). In recent research conducted in an integrated poultry farm in Ecuador, 4.1% (8/194) of samples were positive for
Salmonella, particularly in animal-based feed (
Villagómez Estrada
et al., 2017). Therefore, the accurate detection of this pathogen in feed is necessary in order to identify the critical points of contamination (facilities, raw materials, transport, storage, producers), that one may apply effective measures to reduce the risk of transmission.
The finding of only
S. Infantis in poultry feed may be attributed to the high level of persistence of this serovar over time in poultry feed, which in turn results in its considerable presence in Ecuadorian broiler chicken (
Andino
et al., 2014;
Vinueza-Burgos
et al., 2016). Although the origin of this serovar in feed it is not well defined, studies have pointed to cross-contamination with feces, persistent contamination of storage bins and surfaces, and poor ingredient selection as main causes of feed contamination with
Salmonella (
Davies & Wray, 1997;
Huss
et al., 2018;
Laban
et al., 2014). Genomic tools, such as multilocus sequence typing, BOX-PCR, (GTG)5-PCR or whole-genome sequencing may help to identify the origin of feed contamination in a larger study.
Drag swabs revealed the presence of two different serovars in the sampled poultry farms: Infantis and Typhimurium. These non-typhoidal
S. enterica serovars are commonly associated with poultry systems and are linked to outbreaks of foodborne illness (
Anderson
et al., 2016;
Pui
et al., 2011). Serovars vary in their persistence over time and geographic distribution around the world (
Hendriksen
et al., 2011), making further studies desirable in order to evaluate the variations in serovar persistence in the locations sampled in this research.
In backyard layer feces sampled at local markets,
S. Infantis alone was detected (2/2), but further evaluation of
Salmonella serovars in backyard flocks is recommended in order to improve surveillance of this potential source of salmonellosis (
Behravesh
et al., 2014). In pooled cloacal swabs, the serovars detected were Infantis (2/7), Typhimurium (2/7) and unidentified serovars (3/7). Infantis and Typhimurium serovars are commonly detected in poultry around the world (
Foley & Lynne, 2008;
Foley
et al., 2011). Complementary analysis by serotyping the unclassified serovars is necessary in order to identify
Salmonella bacteria not covered by the panel.
Data remains insufficient concerning the prevalence of
Salmonella in poultry systems in Ecuador and in Latin America in general. The coordination of similar future studies may provide a starting point for surveillance of zoonotic bacteria within a defined public health area, leading to an improvement in policies and safe practices in the food industry. Such studies may reduce the risk of infection and establish protocols for corrective measures to be implemented in key upstream points of the chain as indicated by the data.
At the same time, the intervention of public health authorities may be required in order to ensure the participation of a fully representative range of poultry businesses in future research. This study depended on the voluntary consent of farm owners to providing samples, which may have led to selection bias. In order to establish a thorough program of surveillance, all potential sources of
Salmonella infection in the region need to be made accessible to researchers.
These findings show a significant presence of
Salmonella in layer farms in the central zone of Ecuador. The predominant serovars are
S. Infantis and
S. Typhimurium, typified by PCR. The principal source of infection could be related with poultry feed. From a public health perspective, it is necessary to establish adequate surveillance of
Salmonella, including protocols covering biosecurity practices, antibiotic usage and random sampling programs.
Data availabilityUnderlying data
Figshare: Dataset 1. Origin, biochemical tests, PCR results and serovar of
Salmonella isolates.
https://doi.org/10.6084/m9.figshare.7726163.v2 (
Calero-Cáceres, 2019a)
Figshare: Dataset 2. PCR results of bcfC, fliC and M.SinI genes.
https://doi.org/10.6084/m9.figshare.7726217.v2 (
Calero-Cáceres, 2019b)
Extended data
Figshare: Supplementary figure 1. Multiplex PCR amplification of serovar-specific genes. a)
bcfC and
mSinI fragments (
S. Infantis), b)
bcfC and
fliC fragments (
S. Typhimurium).
Data are available under the terms of the
Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
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885267110.1111/j.1365-2672.1996.tb03216.x10.5256/f1000research.19945.r45085Reviewer response for version 1Gonzalez-GustavsonEloy1Refereehttps://orcid.org/0000-0001-7328-2983
Facultad de Medicina Veterinaria, Universidad Nacional Mayor de San Marcos, Lima, Peru
Competing interests: No competing interests were disclosed.
The scientific names in the manuscript need to be reviewed.
The author use incorrectly the terms prevalence and incidence as synonyms.
Incidence is defined as the presence of new cases of an illness over a period of time. The manuscript does not correspond to an incidence study.
I would like to see more information in the manuscript about the design to consider the results as prevalence. I have not found details about the representativeness of the sampling (sample size to get a prevalence) or if they were obtained randomly. If the method does not satisfy the requirement to be considered as prevalence, I recommend replacing the term prevalence by frequency, presence or proportion.
The confidence interval in one case was incorrectly estimated.
"Feces from backyard layers showed the presence of
Salmonella in 2 of 21 samples (10%, 95%CI: 0–22)"
This values have to be calculated based on binomial distribution. Actually in the manuscript, the confidence interval was estimated based on normal or t distribution and 0 was included as lower interval.
Is the work clearly and accurately presented and does it cite the current literature?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
10.5256/f1000research.19945.r45084Reviewer response for version 1Gaviria CantínTania1Referee
Departamento de Ciencias Naturales, Exactas y Estadística. Facultad de Ciencias Básicas, Universidad de Santiago de Cali, Cali, Colombia
Competing interests: No competing interests were disclosed.
I consider that the title is not totally in agreement with the study. This could be modified at “
Samonella diversity isolated from ... “ without the word “Prevalence” because the study is not a follow-up of previously reported cases in those same conditions
Introduction
In the fourth line you would change the word “poisoning” by contamination
At the end correct one “and” that is in italics
When you say "among the layer hens, no information about prevalence or diversity of
Salmonella has been reported", you could specify or support if that lack of information is only in Ecuador or at a South American or global level. In the same way, it could important to include some epidemiological data of salmonelosis in Ecuador.
Methodology
I think that is not necessary to do DNA extraction to perform PCR, because
Salmonella an is gram negative bacteria and during the start of the PCR where the temperature rises to 90 ° C it is possible to destroy cellular wall and release the DNA. However, it does not affect the results, taking into account that the procedure has been carried out correctly avoiding cross contamination.
On the other hand, in order to reproduce the methodology of this study, the program used for PCR should be indicated. The concentration of primers (30 uM) is the final or initial concentration?
It would be useful to indicate why those particular genes were used as molecular markers for the identification of the species and the serovars
Results
You can ignore the phrase "amplification of the bcfC gene" and leave the table 1 referenced because it is a bit redundant
Regarding statistical analysis and sample number in representation or not, I prefer not to give many opinions because I'm not qualified enough for that. However, it would be important that the methodology explain in detail how the statistical analysis was performed and the calculation of the incidence.
In order of reference, table 1 of results should be table 2 and vice versa
Regarding table 1 (positive S
almonella samples ...), it could be a little more consistent with what they explain in the methodology because it took me a bit to understand it, taking into account the classification of columns in terms of location, samples, etc.
Discussion
I believe that at some point it can be said that the regulations of Ecuador in relation to the health and control of layer farms and local markets and explain how the work is contributing to improve local control, taking into account that this layer farms produce around 60% of egg production in the country.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.