Presence and diversity of Salmonella isolated from layer farms in central Ecuador

Samples

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 Salmonella

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 1). One virulence gene related with fimbrial cluster (bcfC) was evaluated as target of S. enterica specie (Zhu et al., 2015). For serovars and biotypes: A modification methylase gene that are specific of S. Infantis (M.SinI) (Ranjbar et al., 2017). 23S rRNA gene associated to S. Typhi (sty) (Pui et al., 2011). A flagellin gene that show specificity to S. Typhimurium (fliC)(Pui et al., 2011). Gallinarum biotypes were identified by steB fimbrial and rhs locus (Zhu et al., 2015). Specific DNA difference fragment (SdfI) served as target to distinguish S. Enteritidis from other serovars (Agron et al., 2001). And a putative membrane protein (gly) to identify S. Kentucky (Zhu et al., 2015). 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 (>10⁷ UFC/ml) diluted in sterile ddH ₂0 was used as template for the PCR reactions. Amplifications were carried out as follows: initial denaturation at 95°C for 1 min; 35 cycles of 95°C for 30 s, annealing temperature according to Table 1 for 30 s, extension at 72°C for 1 min; and a final elongation step at 72°C for 7 min. 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.

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 stock), 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).

Assessing the presence of Salmonella

Overall, 31 out of 34 isolates which showed phenotypic characteristics in accordance with Salmonella were confirmed by PCR as S. enterica (Table 2). Of the 21 farms, 16 (76%, 95%CI: 53-92) showed the presence of Salmonella on environmental surfaces, and Salmonella bacteria were isolated in 7 pooled cloacal swabs (33%, 95%CI: 15–57) and in 6 of 18 composite feed samples (33%, 95%CI: 13–59). Feces from backyard layers showed the presence of Salmonella in 2 of 21 samples (10%, 95%CI: 1–30). 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).

Figure 1. Distribution of Salmonella serovars according to the evaluated matrices.

Underlying 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).

https://doi.org/10.6084/m9.figshare.7732934.v1 (Calero-Cáceres, 2019c.)

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).