Bataller et al. BMC Veterinary Research (2020) 16:268 https://doi.org/10.1186/s12917-020-02492-3 RESEARCH ARTICLE Open Access Dogs as a source of Salmonella spp. in apparently healthy dogs in the Valencia Region. Could it be related with intestinal lactic acid bacteria? E. Bataller1*, E. García-Romero2, L. Llobat1, V. Lizana3,4 and E. Jiménez-Trigos1* Abstract Background: Although salmonellosis is considered one of the most important food-borne zoonotic diseases in Europe, close contact between dogs and their owners can also be a potential source of Salmonella spp. for humans. This study assessed the prevalence and antimicrobial resistance of Salmonella spp. in apparently healthy dogs in the Valencian Region, eastern Spain. Moreover, a macroscopic comparison of lactic acid bacteria in both Salmonella-positive and Salmonella-negative dogs was carried out. Results: Of a total of 325 dogs sampled, 6 (1.85%) were positive for Salmonella spp. with 3 different serotypes, Havana (3), Mikawasima (2) and monophasic Typhimurium (1). All isolates were susceptible to all antimicrobials tested except monophasic S. Typhimurium, which was resistant to ampicillin. Finally, macroscopic results revealed that lactic acid bacteria had higher heterogeneity in the Salmonella-negative dogs than in the Salmonella-positive dogs. Although the results in our study showed a low prevalence of Salmonella spp., raw food has been suggested as a risk factor for bacteria in dog faeces. Conclusions: Public awareness campaigns on good hygiene practices, especially after handling canine faeces or raw food, are necessary. Furthermore, to reduce the potential transmission of bacteria, dogs should be fed food that has been properly cooked, as raw or undercooked food can be a source of zoonotic pathogens. Moreover, further studies must be performed to determine the relationship between lactic acid bacteria and Salmonella spp. in dog faeces. Keywords: Salmonella, Dog, Prevalence, Zoonosis, Risk factors, Raw food Background meat and meat products contaminated with bacteria Salmonellosis is considered one of the most important were the main sources of human infection [1]. However, foodborne zoonotic diseases in Europe [1]. In 2017, 91, companion animals can also be a source of Salmonella 662 confirmed human salmonellosis cases were reported spp. for humans [2–6]. by all state members in the EU, and eggs, egg products, Dogs are one of the most important companion ani- mals in the world [7]. It has been estimated that in Spain, there are more than 7 million pet dogs [8]. Close * Correspondence: esther.bataller@uchceu.es; estrella.jimenez@uchceu.es 1 Research Group Microbiological Agents Associated with Animal contact between dogs and humans has been recognized Reproduction (PROVAGINBIO), Department of Animal Production and Health, as a potential source of Salmonella spp. zoonotic infec- Veterinary Public Health and Food Science and Technology (PASAPTA), tion [2, 4, 9–11]. In fact, the most frequent serotypes Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Carrer Tirant lo Blanc 7, 46115 Alfara del Patriarca, València, Spain isolated from human gastroenteritis cases correspond Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bataller et al. BMC Veterinary Research (2020) 16:268 Page 2 of 8 with more prevalent serotypes in dogs [11, 12]. In Rectal swabs were obtained from 85 dogs from house- addition, companion animals could be an important holds (26.2%), 84 dogs from animal shelters (25.8%), and source of multidrug-resistant Salmonella spp. [13]. 156 dogs from hunting kennels (48.0%). Of the 325 sam- Therefore, pet dogs should be considered a public health pled dogs, 140 (43.1%) and 185 (56.9%) were males and risk [4]. Children, elderly and immunocompromised in- females, and 49 (15.1%) and 276 (84.9%) were puppies dividuals have a higher risk of salmonellosis because and adult dogs, respectively. they are more susceptible to infection [2, 11, 14, 15]. Regarding feeding, of the 325 dogs analysed, 196 ate Since the 1970s, previous studies have reported the commercial pet food (60.3%), and the remaining dogs zoonotic transmission of Salmonella spp. from dogs were also fed home-prepared food (39.7%). Moreover, 36 [16]. Nevertheless, the prevalence of this bacterium in dogs (11.1%) were fed raw food. The type of water dogs varies considerably. First, dogs usually act as source was also asked in the questionnaire; 11 dogs asymptomatic carriers, and they can shed one or more (3.4%) drank bottled water, while 269 (82.8%) drank run- serotypes intermittently for more than 6 weeks [11, 17]. ning water and 45 (13.8%) drank from a water well. Fi- Moreover, dogs can harbour Salmonella spp. in the in- nally, whether the dogs were in contact with other testine and mesenteric lymph nodes without evidence of animal species was also asked in the questionnaire, and clinical signs [10, 11, 18]. Different studies have shown a 178 dogs of 325 (54.8%) were in contact with wild ani- wide prevalence of Salmonella spp. that oscillates be- mals. Independent of the environment where dogs lived, tween 0 and 79% [11, 12, 18–25]. This variability makes only 6 (1.85%) of the 325 rectal swabs analysed were establishing the real prevalence among dog populations positive for Salmonella spp. No significant differences a challenge. were found between the percentage of Salmonella spp. Other factors that have been reported to influence the and the age, sex, type of food, water source or contact prevalence of Salmonella spp. in dogs are the environ- with wild animals. However, the prevalence of Salmon- ment where animals live or contact with wild animals or ella spp. was higher in animals that were fed raw food other infected animals [11, 18, 26]. Additionally, animal compared to those that did not eat raw food, and this feeding has been described as one of the main risk fac- difference was statistically significant (p value ≤0.01). tors for the prevalence of Salmonella spp. in dogs [11]. Serotypes isolated from dog rectal samples are also Feeding dogs with raw food has also been related to the shown in Table 1. Serotyping revealed 3 different sero- high prevalence of salmonellosis [12, 20, 21, 27, 28]. vars: S. Mikawasima (n = 2); S. Havana (n = 3) and Another factor related to the increase in Salmonella Monophasic S. Typhimurium (n = 1). spp. in animals could be associated with the alteration of normal microbiota, which protects the gastrointestinal Antimicrobial susceptibility of Salmonella isolates tract from colonization by pathogens; alterations in the The antimicrobial susceptibility of Salmonella serotypes normal microbiota could provide a suitable environment is shown in Table 2. All serotypes were susceptible to all for bacterial replication [29, 30]. antimicrobials tested except monophasic S. Typhimur- As mentioned above, the prevalence of Salmonella se- ium, which was resistant to ampicillin. rotypes in clinically healthy dogs varies notably and may even be different depending on the country [11]. As far Macroscopic LAB differences between Salmonella-positive as we know, no studies on the prevalence of Salmonella and Salmonella-negative dogs spp. in dogs have been performed in Spain. Therefore, LAB from Salmonella-negative dogs (n = 319) and Sal- the objective of this study was to determine the presence monella-positive dogs (n = 6) were grown on MRS agar. of Salmonella spp. in asymptomatic dogs housed in dif- Macroscopic analysis was carried out, and subjective dif- ferent environmental conditions in the Valencian Region ferences were observed. Figures 1 and 2 show the macro- (southern Spain). Moreover, the antimicrobial drug sus- scopic profile of LAB from the Salmonella-negative and ceptibility of the isolates was determined, and macro- Salmonella-positive dogs, respectively. As an initial ap- scopic analysis of gastrointestinal lactic acid bacteria proximation, Salmonella-negative animals seemed to have (LAB) in Salmonella spp.-positive and Salmonella spp.- more LAB than Salmonella-positive animals. negative animals was also evaluated. Discussion Results Although dogs may not play as large of a role in the The prevalence of Salmonella spp. in apparently healthy transmission of salmonellosis to people as food, dogs dogs have long been known to be asymptomatic carriers of The number of dogs sampled and the prevalence of Sal- Salmonella serotypes [31]. Their close relationship and monella spp. is shown in Table 1. A total of 325 dogs frequent contact with humans, especially children, may housed in three different environments were examined. make these animals a potential source of Salmonella Bataller et al. BMC Veterinary Research (2020) 16:268 Page 3 of 8 Table 1 The prevalence of Salmonella spp. in apparently healthy dogs and variables included in the study Variable Categories No. of Dogs No. positive for Salmonella Serotype (n) X2 (p-value) (%) Salmonella spp. Dog population Households 85 (26.2) 1 S. Mikawasima (1) 1.86 (0.39) Animal 84 (25.8) 3 S. Havana (3) shelters Hunting 156 (48) 2 S. Mikawasima (1) Monophasic S. Typhimurium (1) kennels Gender Males 140 (43.1) 3 S. Mikawasima (1) S. Havana (1) Monophasic S. 0.12 (0.73) Typhimurium (1) Females 185 (56.9) 3 S. Mikawasima (1) S. Havana (2) Age Puppies 49 (15.1) 2 S. Havana (2) 1.59 (0.21) Adults 276 (84.9) 4 S. Mikawasima (2) S. Havana (1) Monophasic S. Typhimurium (1) Diet Commercial 196 (60.3) 3 S. Havana (3) 0.27 (0.60) Home 129 (39.7) 3 S. Mikawasima (2) Monophasic S. Typhimurium (1) prepared Raw food Yes 36 (11.1) 4 S. Havana (3) Monophasic S. Typhimurium (1) 19.18 (< 0.01)* consumption No 289 (88.9) 2 S. Mikawasima (2) Source of water Bottled water 11 (3.4) 0 – 0.24 (0.89) Running 269 (82.2) 5 S. Mikawasima (1) S. Havana (3) Monophasic S. water Typhimurium (1) Well water 45 (13.8) 1 S. Mikawasima (1) Contact with wild Yes 178 (54.8) 2 S. Mikawasima (1) Monophasic S. Typhimurium (1) 1.13 (0.29) animals No 147 (45.2) 4 S. Mikawasima (1) S. Havana (3) Overall 325 (100) 6 *p value ≤0.01 was considered significant spp. for humans and thereby represent an important dogs were sampled only once. The low prevalence ob- public health issue [2, 10, 11, 18]. served in our study could be related to the fact that only The isolation of Salmonella spp. from clinically healthy one faecal sample was collected, as intermittent shedding dogs has been reported to be between 0 and 79% [11, of Salmonella spp. has been well documented [11, 17]. 21, 23, 24, 27]. The results obtained in the present study However, rectal swab samples were employed in other showed a prevalence of Salmonella spp. of 1.8% in ap- studies to determine the presence of Salmonella in dog parently healthy dogs in Valencian Region (Southern faeces; these studies observed similar results and a Spain). Our results were in accordance with the litera- higher prevalence of Salmonella [32, 33]. ture. In most studies, the prevalence of Salmonella-posi- The prevalence of Salmonella spp. in dogs has also tive dogs was below 3% [12]. Nevertheless, previous been reported to depend on the immediate environment studies have shown that the prevalence of Salmonella in in which the animals live [11]. For example, the rates of asymptomatic dogs can vary geographically [10, 11, 18, isolating Salmonella from stray dogs have been reported 19, 22, 23, 25]. Factors associated with these variations to be significantly higher than those from household could be in part related to differences in the sample dogs [32]. In the current study, samples were collected sizes, faecal sampling conditions and isolation and detec- from dogs housed in three different environments: tion methods employed [11, 23]. In the present study, households, animal shelters and hunting kennels. The Table 2 Antimicrobial resistance of Salmonella spp. isolated from dog rectal samples Salmonella serotype (No. of Antimicrobial susceptibility isolates) AMP CTX CAZ GM ND CIP AZM TGC SXT CT C S. Havana (3) S S S S S S S S S S S S. Mikawasima (2) S S S S S S S S S S S Monophasic S. Typhimurium (1) R S S S S S S S S S S AMP Ampicillin, CTX Cefotaxime, CAZ Ceftazidime, GM Gentamicin, ND Nalidixic acid, CIP Ciprofloxacin, AZM Azithromycin, TGC Tigecycline, SXT Trimethoprim- sulfamethoxazole, CT Colistin, C Chloramphenicol, S Susceptible, R Resistant Bataller et al. BMC Veterinary Research (2020) 16:268 Page 4 of 8 Fig. 1 Macroscopic image of LAB from Salmonella spp.-negative dogs on MRS agar plates after 48 h of incubation at 37 °C under anaerobic conditions Fig. 2 Macroscopic image of LAB from Salmonella spp.-positive dogs on MRS agar plates after 48 h of incubation at 37 °C under anaerobic conditions Bataller et al. BMC Veterinary Research (2020) 16:268 Page 5 of 8 results of our study are in accordance with Reimschues- Other authors have reported the isolation of resistant sel et al. (2017) [12]. They found that there was no sig- Salmonella strains from dogs [11, 12, 32, 52]. In nificant statistical association with Salmonella status and addition, adding raw animal products to pet dog diets living or exposure with other animals, age, exposure to has been identified as a risk factor for the presence of water sources, hunting or performing sport activities. antimicrobial-resistant Salmonella spp. [11, 53]. However, different studies have shown that the preva- New strategies are urgently needed to manage anti- lence of Salmonella in dogs exposed to contaminated microbial resistant infections [34, 48, 53]. Among the environments including hunting dogs or stray dogs is various methods to reduce the use of antibiotics [54], higher than that in household dogs [12, 23, 32, 33]; this probiotics are expected to be an alternative interven- result could be because dogs are more likely to roam tion measure to prevent bacterial infection in dogs free, scavenge, be in contact with carcasses or offals of [55]. To identify beneficial probiotic bacteria that in- wildlife and be fed raw or undercooked food [23, 33, 34]. hibit pathogens, a better understanding of the dog As mentioned, differences in the prevalence of Sal- microbiome is needed [56–58]. As a preliminary ap- monella can also vary depending on feeding practices proach, we compared LAB in samples from both Sal- [11]. Feeding pet raw meat-based diets (RMBDs) has re- monella spp.-positive and Salmonella spp.-negative ceived increasing attention in recent years [35]. Our re- animals, and it seems that macroscopic differences sults showed that 4 of 6 dogs positive for Salmonella were observed. Commensal bacteria maintain the sta- were fed raw poultry carcasses. These results are in ac- bility of the digestive tract and can help prevent in- cordance with other studies that concluded that con- testinal infections by modulating the immune suming raw diets increases the risk of carrying response and inhibiting certain pathogens such as Salmonella spp. [12, 20, 21, 24, 27, 36]. Salmonella spp. that cause infections [58, 59]. The Different studies have recorded that serovars found subjective differences observed at the macroscopic more frequently in dog faeces are similar to those found level in our study could suggest that some LAB may in humans [10, 12, 22, 32, 36, 37]. In the present study, protect against the pathogen or, on the other hand, three serotypes were isolated, monophasic Salmonella the presence of Salmonella spp. in dogs could inhibit Typhimurium (16.7%), S. Havana (50%) and S. Mikawa- beneficial LAB strains. Our preliminary results could sima (33.3%), which have also been implicated in human be in accordance with other studies that observed salmonellosis [38–40]. All serotypes have been reported that gut LAB in pigs inhibit the growth of Salmonella previously in dogs and humans [1, 16, 18, 39–45], and and prevent the adhesion of bacteria in the intestinal they have also been reported to be present in raw food tract [60]. [1, 39]. In that sense, further studies must be carried out to In our study, S. Havana were found in the faeces of address the limitations of our study and analyse whether Salmonella-positive dogs fed raw chicken carcasses. This these differences could be related to Salmonella spp. in- serotype has been isolated previously from poultry prod- fection. It would be interesting to identify whether some ucts [46]. Pace et al. [38] determined a possible human species of LAB in Salmonella spp.-negative dogs are able infection caused by S. Havana from dog food contamin- to inhibit Salmonella spp., which could explain the ab- ation. S. Mikawasima has also been found in both wild sence of infection. Host species specificity is considered boar from cattle-free and cattle-grazed areas in Spain a requirement for probiotics, especially as a therapy for [47], and in our study, S. Mikawasima was isolated from GI disorders [58]. In that sense, the study of dog micro- one hunting dog in contact with these species. More- biota may lead to the discovery of new therapies that over, there has been an unusual increase in the number can act against pathogens such as Salmonella spp. of S. Mikawasima infections in humans [39, 44]. There- fore, these results underline the potential public health Conclusion risk of dog- and pet-contaminated foods [12, 22, 34]. In conclusion, the results obtained in this study indicate In addition, a major problem in terms of both animal that apparently healthy dogs can act as subclinical car- and human health is the emergence and increase in anti- riers of Salmonella spp. in the Valencian region, eastern biotic resistance [34]. In fact, Salmonella has been listed Spain. This could be significant to public health, as dogs by the World Health Organization (WHO) as one of the are in close contact with humans. However, further antibiotic-resistant priority pathogens and has become a studies must be performed because it is known that the worldwide health issue [48]. The results obtained in the shedding of Salmonella in dog faeces is influenced by present study showed that all serotypes isolated in our several factors, such as diet, sampling procedures, and study were susceptible to all antibiotics tested. This is in geographic area. Moreover, additional studies must be accordance with other studies [11, 12, 49–51]. Only performed to determine the relationship between LAB monophasic S. Typhimurium was resistant to ampicillin. and Salmonella spp. in dog faeces. Bataller et al. BMC Veterinary Research (2020) 16:268 Page 6 of 8 Methods (30 μg), ciprofloxacin (5 μg), azithromycin (15 μg), tige- Sample collection and questionnaire cycline (15 μg), trimethoprim-sulfamethoxazole (25 μg), All animals were handled according to the principles of colistin (10 μg) and chloramphenicol (5 μg). Antimicro- animal care published by Spanish Royal Decree 53/2013 bial susceptibility was tested according the European [61]. Sample collection was carried out in the Valencian Committee on Antimicrobial Susceptibility Testing Region (eastern Spain) between October 2017 and June (EUCAST) guidelines [64]. The source for zone diame- 2018. A total of 325 healthy dogs housed in three differ- ters used for interpretation of the test was http://www. ent environments were sampled (dogs were housed in eucast.org/clinical_breakpoints/. Zone diameters were households or animal shelters or were hunting dogs). interpreted and categorized as susceptible, intermediate Rectal specimens were collected using sterile cotton or resistant according to the EUCAST clinical break- swabs (Cary Blair sterile transport swabs, Deltalab, Bar- point tables. celona, Spain) by rotating the swab inside the rectum of the dog and then transported under refrigeration to the Lactic acid bacteria isolation Laboratory of “Agentes microbiológicos asociados a la LAB isolation was carried out using the same rectal sam- Reproducción Animal-ProVaginBIO”, UCH-CEU Uni- ples collected for Salmonella spp. isolation. Swabs were versity, for Salmonella spp. isolation. homogenized in BPW, and 100 μl was inoculated in the Data from each dog were also collected by a question- medium used for identification of LAB, especially Lacto- naire developed for this study (Additional file 1) to de- bacillus Man, Rogosa and Sharpe (MRS agar) (Scharlab, termine the possible risk factors for Salmonella spp. Barcelona, Spain) [65, 66], and incubated for 24–48 h infection, especially those related to the environment under anaerobic conditions. After incubation, subjective where animals were housed, the diet or type of food, macroscopic observation was carried out. contact with other animal species and the source of water. Other data were also included in this question- Statistical analysis naire, including gender and age. All questionnaires were Statistical analysis was performed with the statistical completed and submitted together with the samples to package R Commander and RcmdrPlugin. The associa- the laboratory. tions between Salmonella occurrence; categorical factors Salmonella spp. isolation, serotyping and determin- were compared using Pearson’s χ2 test, and the confi- ation of antibiotic susceptibility. dence intervals for prevalence estimates were calculated Salmonella isolation was performed according to ISO using the Wilson score interval method. A p-value < 6579: 2002 (Annex D) [62]. First, samples were preen- 0.01 was reported as statistically significant. riched for 18 ± 2 h at 37 °C ± 1 °C in 1:10 vol/vol buffered peptone water 2.5% (BPW; Scharlau, Barcelona, Spain). Then, 0.1 mL of the preenriched sample was transferred Supplementary information Supplementary information accompanies this paper at https://doi.org/10. onto Semi-Solid Modification Rappaport Vassiliadis agar 1186/s12917-020-02492-3. plates (MSRV; Difco, Valencia, Spain) and incubated at 41.5 °C ± 1 °C for 24–48 h. Suspicious growth on these Additional file 1. Questionnaire plates was selected for inoculation onto Xylose–Lysine– Deoxycholate (XLD; Liofilchem, Valencia, Spain) and Abbreviations ASAP (ASAP, bioMerieux, Madrid, Spain) agar plates, ASAP: Chromogenic Culture Media for Rapid Salmonella Detection; which were incubated at 37 °C ± 1 °C for 24–48 h. After BPW: Buffered Peptone Water; LAB: Lactic Acid Bacteria; MRS: Man Rogosa Sharpe; MSRV: Semisolid Modification Rappaport Vassiliadis; XLD: Xylose– the incubation period, presumptive Salmonella colonies Lysine–Deoxycholate were selected, streaked onto nutrient agar plates (Schar- lab, Barcelona, Spain) and incubated at 37 °C ± 1 °C for Acknowledgements 24 ± 3 h. Then, a biochemical test using API (API-20, We would like to thank the animal shelters and dog owners for their cooperation during sample collection. bioMerieux, Madrid, Spain) was performed to confirm the presence of Salmonella spp. Moreover, Salmonella Authors’ contributions isolates were serotyped by the Ministry of Agriculture, EJT and EB contributed to the study design, laboratory investigation and Fisheries and Food Reference Laboratory (Algete, preparation of the draft manuscript. EGR provided technical assistance during laboratory investigation. VL was involved in sample collection. LLl Madrid, Spain) according to the Kauffman-White-Le participated in the data analysis. All authors read and approved the final Minor scheme. manuscript. The antibiotics selected to test Salmonella spp. anti- microbial susceptibility were those set forth in Decision Funding This work was funded by the precompetitive projects CEU – Banco 2013/653 [63]: ampicillin (10 μg), cefotaxime (30 μg), cef- santander (FUSP-BS-PPC15/2016). The funding agency was not involved in tazidime (30 μg), gentamicin (10 μg), nalidixic acid design of study, data collection, analysis of data and manuscript writing. Bataller et al. BMC Veterinary Research (2020) 16:268 Page 7 of 8 Availability of data and materials 10. Leonard F. Salmonella infection and carriage: the importance of dogs and The datasets analysed during this study are available from the corresponding their owners. Vet Rec. 2014;174:92–3. author on reasonable request. 11. Lowden P, Wallis C, Gee N, Hilton A. Investigating the prevalence of Salmonella in dogs within the midlands region of the United Kingdom. Ethics approval and consent to participate BMC Vet Res. 2015;17:11–239. This work was approved by the precompetitive projects CEU – Banco 12. Reimschuessel R, Grabenstein M, Guag J, Nemser SM, Song K, Qiu J, Clothier Santander (FUSP-BS-PPC15/2016). All animals were handled according to the KA, Byrne BA, Marks SL, Cadmus K, Pabilonia K, Sanchez S, Rajeev S, Ensley S, principles of animal care published by Spanish Royal Decree 53/2013 (Official Frana TS, Jergens AE, Chappell KH, Thakur S, Byrum B, Cui J, Zhang Y, Spanish State Gazette [BOE], 2013). Sampled collection was approved by the Erdman MM, Rankin SC, Daly R, Das S, Ruesch L, Lawhon SD, Zhang S, Ethics Committee and Animal Experimentation of UCH-CEU University. In- Baszler T, Diaz-Campos D, Hartmann F, Okwumabua O. Multilaboratory formed oral consent, also approved by the Ethics Committee and Animal Ex- survey to evaluate Salmonella prevalence in diarrheic and nondiarrheic dogs perimentation of the UCH-CEU University, was obtained from the dog and cats in the United States between 2012 and 2014. 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