Salmonella spp. contamination in commercial layer hen farms using different types of samples and detection methods M. C. Soria,∗,1 M. A. Soria,∗ D. J. Bueno,∗,3 E. I. Godano,† S. C. G´omez,‡,2 I. A. ViaButron,∗ V. M. Padin,§ and A. D. Rog´e§ ∗Instituto Nacional de Tecnolog´ıa Agropecuaria EEA Concepci´on del Uruguay, Ruta Provincial 39 Km 143,5, 3260, Concepci´on del Uruguay, Entre R´ıos, Argentina; †Tecnovo S.A., Parque Industrial C.C. 17, 3116, Crespo, Entre R´ıos, Argentina.; ‡Fundaci´on ArgenINTA, Ruta 11, Km 12.5, 3101, Oro Verde, Paran´a, Entre R´ıos, Argentina; and §Servicio de Ant´ıgenos y Antisueros, Instituto Nacional de Producci´on de Biol´ogicos (INPB) - ANLIS “Dr Carlos G. Malbr´an”, Av. V´elez Sarsfield 563, C1282AFF, Ciudad de Buenos Aires, Argentina ABSTRACT The performance of detection methods Agreement was between fair to good depending on the (culture methods and polymerase chain reaction as- sample, and it was good between isolation and PCR (fe- say) and plating media used in the same type of sam- ces and boot-swabs), without agreement for feed sam- ples were determined as well as the specificity of PCR ples. Salmonella spp. PCR was positive for all strains, primers to detected Salmonella spp. contamination in while S. Typhimurium PCR was negative. Salmonella layer hen farms. Also, the association of farm character- Enteritidis PCR used was not specific. Based in the istics with Salmonella presence was evaluated. Environ- multiple logistic regression analyses, categorization by mental samples (feces, feed, drinking water, air, boot- counties was significant for Salmonella spp. presence swabs) and eggs were taken from 40 layer hen houses. (P-value = 0.010). This study shows the importance Salmonella spp. was most detected in boot-swabs taken of considering different types of samples, plating media around the houses (30% and 35% by isolation and PCR, and detection methods during a Salmonella spp. moni- respectively) follow by fecal samples (15.2% and 13.6% toring study. In addition, it is important to incorporate by isolation and PCR, respectively). Eggs, drinking wa- the sampling of floors around the layer hen houses to ter, and air samples were negative for Salmonella de- learn if biosecurity measures should be strengthened tection. Salmonella Schwarzengrund and S. Enteritidis to minimize the entry and spread of Salmonella in the were the most isolated serotypes. For plating media, rel- houses. Also, the performance of some PCR methods ative specificity was 1, and the relative sensitivity was and S. Enteritidis PCR should be improved, and biose- greater for EF-18 agar than XLDT agar in feed and curity measures in hen farms must be reinforced in the fecal samples. However, relative sensitivity was greater region of more concentrated layer hen houses to reduce in XLDT agar than EF-18 agar for boot-swab samples. the probability of Salmonella spp. presence. Key words: Salmonella, detection, sampling, layer hen farm 2017 Poultry Science 00:1–11 http://dx.doi.org/10.3382/ps/pex053 INTRODUCTION range of food products, poultry meat and eggs are among the most frequently implicated sources of human The intense international trade of animals and animal Salmonella outbreaks (Gast, 2013). Therefore, knowl- products facilitates the spread of Salmonella spp., mak- edge of the actual prevalence of Salmonella spp. in ing salmonellosis an international public-health sub- laying hens and the factors that influence the pres- ject, responsible for serious economic losses to the poul- ence and persistence of Salmonella on a layer hen try industry and governments. Although humans can farm are of the utmost importance (Van Hoorebecke become infected by Salmonella spp. through a wide et al., 2010b). Because Salmonella Enteritidis (SE) and other im- portant serotypes do not typically cause mortality in C 2017 Poultry Science Association Inc. layer flocks, samples collected from the environment Received October 12, 2016. Accepted March 2, 2017. tend to more readily indicate the presence of them in 1Present address: INIQUI-CONICET, Universidad Nacional de the flock (Holt et al., 2011). Environmental monitoring Salta, Avda. Bolivia 5150, C.P. 4400, Salta, Argentina. is a useful, effective and less invasive method to predict 2 Present address: Facultad de Ciencia y Tecnolog´ıa, Universidad potential infection or colonization of the poultry flocks Aut´onoma de Entre R´ıos, 25 de Mayo 353, 3260, Concepci´on del Uruguay, Entre R´ıos, Argentina. (Waltman and Gast, 2008). Furthermore, a high level 3Corresponding author: [email protected] of environmental contamination of the house is also 1 2 SORIA ET AL. associated with a higher risk of producing contaminated MATERIALS AND METHODS eggs in laying houses (Henzler et al., 1998). Even after cleaning and disinfection among laying cycles, SE can Sampling appear in a variety of environmental samples (Dewaele et al., 2012). Forty layer hen houses belonging to 30 hen farms A wide range of culture methods and polymerase from 6 counties (Colon, Diamante, Gualeguay, Nogoy´a, chain reaction (PCR) assays are available, and sev- Paran´a, and Uruguay) of Entre Rios, Argentina were eral studies had been developed to test their ability to sampled one time, from August 2011 to April 2012. At detect Salmonella in different matrices (Rybolt et al., the moment of sampling, 39 layer hen flocks, housed 2004; Myint et al., 2006; Eriksson and Aspan, 2007; in battery cage systems, were in egg-laying phase. One Love and Rostagno, 2008; Singer et al., 2009). Rou- flock was 4 months old. Without this flock, the average tinely used methods for isolating Salmonella rely on age of the hens was 18 months, with 7 and 36 months as pre-enrichment in nonselective media, selective enrich- minimum and maximum, respectively. Flock sizes were ment, and plating on selective and differential media. between 450 and 30,500 hens. Most of the layer hens Subsequently, the pathogens are identified by biochem- were white egg layers, and they had not received any ical and serological tests. According to Rybolt et al. Salmonella vaccine. (2004), the sensitivity and specificity of the method Automatic and manual battery cage systems for layer depend on the sample type as well as the isolation hens were considered in the sampling. Automatic sys- conditions. Also, the process of isolating Salmonella tems included manure belts, mobile feed chutes, mecha- spp. for environmental monitoring is determinate by nized egg collections, and advanced climate systems for different factors: sampling, level of contamination by cooling and ventilation. On the other hand, the manual the pathogen, or presence of competitors. Depending battery cage farms did not have automated infrastruc- on the type of competitive bacteria, detection of occa- ture and most of the activities were performed manually sional colonies of Salmonella may be easier if the ap- inside the house. propriate plating medium has been used (Busse, 1995). When the farm had 4 or fewer houses, one commer- In addition, processing a greater number of samples cial layer hen house was sampled. If the farm had more with a single effective method is probably more ef- than 4 houses, 2 houses were sampled. When there were ficient than testing a small number of samples us- both manual battery cage and automatic systems on ing complex laboratory protocols (Carrique-Mas and the same farm, one of each type of house was sampled. Davies, 2008). So, 33 and 7 manual and automatic battery cage houses The diversity of sources from which salmonellae can were sampled, respectively. If the farm had animals with be introduced into flocks or houses complicates efforts different ages, the houses that had older layer hens were to establish specific critical control points for preventing selected for the study because they are more likely to zoonotic Salmonella infections in poultry (Gast, 2013). be contaminated with Salmonella spp. (Van Hoorebeke In addition, every poultry farm has its own risk pro- et al., 2010a). file for the introduction of pathogens, subsequent de- velopment of disease, and the spread of pathogens to other farms. This risk profile is determined by a com- plex interaction between the levels of infection in an Number and Sample Types Taken area, the measures implemented on the farm to pre- on Layer Hen Farms vent disease, and other factors including the density of farms in the area and linkages with other farms Feed. Samples were taken from each mobile feed and markets (Sims, 2007). Different countries have dif- chute in manual battery cage houses. In houses without ferent regulations to control Salmonella spp. in lay- any kind of chute, samples were collected from layer ing flocks. Most of them are focused on Salmonella feeders or from the external silo. In automatic house Typhimurium (ST) and SE (Barroso, 2011;USDA, systems, samples were taken from mobile feed chutes. 2011). In all cases, 3 ± 1 kg of feed were taken and put in Minimizing Salmonella contamination in commercial plastic bags, using disinfected plastic spoons. layer hen houses required a continuous improvement of Eggs. Two fiber cartons of 30 eggs each one were the environmental monitoring process (Im et al., 2015), collected from each house. Mainly, dirty eggs were se- methods used for pathogen detection, and the manage- lected to be sampled. It was not possible to analyze ment measures taken in poultry production. For this eggs from 2 layer houses from different farms. In one of reason, the aims of this work were to 1) estimate the them, the hens were not in laying phase. From the other contamination of Salmonella spp. in layer hens farms house, the farm owner did not authorize egg sampling. using different types of samples, 2) evaluate the perfor- Feces. Pooled feces (300 ± 50 g) were taken from all mance of detection methods, plating media used and belts in each line of the house after running the manure the specificity of PCR primers, and 3) study the asso- removal system (automatic).