Multi-locus sequence typing of Salmonella enterica isolates from dog treats and raw meat-based dog food in Japan

Abstract

Objective

Salmonella is an important zoonotic foodborne pathogen which is recognized as a major public health concern worldwide. Salmonella contamination are highly prevalent in dog treats and raw meat-based dog food (RMBDs). And dog treats and raw meat-based dog food are often implicated as main sources of human infection. Many epidemiological studies have focused on the characteristics of Salmonella in many countries. But there are no such reports in Japan. This study was conducted to investigate the genetic characteristics of Salmonella using multi-locus sequence typing (MLST).

Results

Fourteen Salmonella isolates were assigned to 8 sequence types: ST19, ST26, ST32, ST34, ST241, ST469, ST864 and ST1861. The most common types were ST32 and ST34 (21%, n = 3). The second most common types were ST26 and ST469 (14%, n = 2).

Introduction

Salmonella is one of the most important foodborne bacterial pathogens in humans and animals [1]. Salmonella is a bacterium that can infect and be isolated from a variety of mammals, birds, reptiles, and insects [1]. Non-typhoidal Salmonella causes gastroenteritis and diarrhea in humans and animals. Salmonella infections tend to cause serious illness in humans, but dogs often show no symptoms. Pet food is known to cause Salmonella infections in humans and dogs. In fact, outbreaks of salmonellosis in humans and dogs have been reported due to pet food [2,3,4]. During the human salmonellosis outbreak in the United States, the genetic characteristics of human-derived Salmonella and pet food-derived Salmonella were compared [2,3,4]. In Japan, Salmonella has actually been detected in pet food [5, 6]. However, genetic characterization of these Salmonella isolates has not been tested. Therefore, we used MLST to analyze the genetic properties of these Salmonella isolates and identified ST types. This study was conducted to investigate their ST types and serotypes.

Main text

Methods

Samples of Salmonella were collected from dog treats and RMBDs in Japan between 2016 and 2017 [5, 6]. A total of fourteen samples were used in this experiment. Seven were from treats and seven from RMBDs. According to the Kauffmann-White scheme, the isolates were serotyped using slide and tube agglutination tests with commercially available antisera (Denka Seiken Co., Ltd., Tokyo, Japan) [7]. In addition, polymerase chain reaction (PCR) was used to serotype the Salmonella isolates [8]. Salmonella DNA was extracted from isolates by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR amplifications were then carried out using primers targeting the gene loci (aroC, dnaN, hemD, hisD, purE, sucA, thrA) described in the previous report. PCR reactions were performed with 10 µL supernatants in a final volume of 50 µL using 2 × GoTaq Master Mix (Promega, Madison, USA), according to the manufacturer’s instructions. Amplification was performed using a LifeECO Thermal Cycler (Hangzhou Bloer Technology Co., Ltd., Zhejiang, China) with the following thermal cycling protocol: initial denaturation (95℃, 2 min) followed by 30 cycles of denaturation (95℃, 1 min), annealing (57℃, 1 min) and extension (72℃, 2 min) with a single final extension (72℃, 5 min). All PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and submitted to Macrogen Japan Corp. (Tokyo, Japan) for sequencing. The resulting DNA sequence data were compared to data in the MLST database (https://pubmlst.org/bigsdb?db=pubmlst_salmonella_seqdef), and all DNA sequence data were submitted to the National Center for Biotechnology Information database GenBank for registration of accession numbers.

Results and discussion

The Salmonella isolates were identified as serotypes S. Infantis (n = 3), S. 4,[5],12:i:- (n = 3), S. Rissen (n = 2), S. Thompson (n = 2), S. Typhimurium (n = 1) and S. Schwarzengrund (n = 1). A total of two isolates could not be serotyped (Tables 1 and 2). S. Infantis, S. Thompson and S. Schwarzengrund were found in human clinical cases [9]. One S. Schwarzengrund isolates was found in RMBDs made from chicken. Since the distribution of S. Schwarzengrund has expanded in Japan, the possibility of S. Schwarzengrund contamination of dog food may increase in the future [10]. Multi-locus sequence typing classification showed that the 14 Salmonella isolates belonged to 8 sequence types (Tables 1 and 2). All DNA sequences were deposited in the National Center for Biotechnology Information database GenBank and assigned accession numbers (Table 3). The most common types were ST32 and ST34 (21%, n = 3). The second most common types were ST26 and ST469 (14%, n = 2). Salmonella ST34 and ST469 have also been found in Korea [11]. Salmonella ST34 has also been detected in pork and humans in Germany [12]. ST19 and ST34 are common ST types in Salmonella Typhimurium infection [13]. ST19 and ST34 are also frequently detected in food animals in Japan [14]. Salmonella ST32 has also been found in dog food in the UK [15]. Salmonella ST32 has been found in humans in Iran [16]. Salmonella ST32 and ST241 have been found in Japanese poultry farms [17]. S. Schwarzengrund ST241 may have become more widespread in Japan [18]. In this way, globally prevalent ST types were detected in Japanese pet food. ST types which have been detected in humans were also found. Salmonella contamination in pet food is a long-standing problem. However, in 2023, an outbreak of human Salmonella infections occurred in the United States due to Salmonella-contaminated dog food [19]. We should continue to carry out these educational activities for pet owners. Pet food manufacturers are required to further improve hygiene management. From the perspective of One Health, the health of dogs, humans and the animals that serve as raw materials, and good hygiene of production environment are important.

Table 1 Sequence types of Salmonella isolates from dog treats between 2016 and 2017 in Japan

Table 2 Sequence types of Salmonella isolates from RMBDs treats between 2016 and 2017 in Japan

Table 3 A List of accession corresponding to each Salmonella gene used in this study

Limitations

In this study, it was not possible to compare the genes with human clinical Salmonella in Japan. In the future, we will compare human clinical strains and pet food-derived strains to further clarify the current situation in Japan.