Commercial Frozen Mice Used by Owners to Feed Reptiles are Highly Externally Contaminated with Salmonella Enteritidis PT8

Introduction

Salmonellosis remains one of the most prevalent zoonoses worldwide (GBD 2017). It has been estimated that nontyphoidal Salmonella causes around 59,153 deaths each year worldwide (Havelaar et al. 2015).

In the United States, more than 1 million cases of foodborne salmonellosis have been estimated to occur annually (Scallan et al. 2011). In Europe, over 80,000 laboratory-confirmed cases are reported each year (EFSA and ECDC 2016). Currently, at a global level, salmonellosis represents an important public health problem, and controlling the disease is an ongoing challenge in most countries (EFSA and ECDC 2016). Although people usually become infected with Salmonella through ingestion of contaminated food, it has been estimated that up to 11% of Salmonella infections overall are acquired from direct or indirect contact with animals, including reptiles (Hale et al. 2012, Cartwright et al. 2016). This attribution is much higher among children, for whom reptile exposure accounts for an estimated 27% of Salmonella infections (Murphy and Oshin 2015).

The popularity and number of exotic reptiles kept as pets have increased in recent years due to reducing cost and increasing availability, leading to a greater number of reptile-associated zoonotic pathogen infections (Schröter et al. 2004, Hernandez et al. 2012, CDC 2014, Cartwright et al. 2016).

Many of these reptiles, especially snakes, are fed on commercially bred mice. These rodents are usually produced in large-scale facilities and distributed in a frozen state nationally and internationally by specialist suppliers (Cartwright et al. 2016). Commercial feeder mice have been previously associated with Salmonella outbreaks (Bertrand et al. 2008, CDC 2014). Nevertheless, the impact of the control measures established after these outbreaks by public health authorities is difficult to evaluate, and reptile-associated salmonellosis (RAS) continues to be reported (Cartwright et al. 2016).

In the United States, the Food and Drug Administration (FDA) code of practice states that commercial feeder rodent producers, suppliers, and distributors should follow the animal food labeling requirements, and all packages of frozen rodents should include safe handling instructions. Despite these control measures, many reptile owners are unaware of the risks associated with exposure to reptiles and feeder mice (Cartwright et al. 2016). When a snake has to be fed, frozen mice are often heated to resemble live food and to make the mice more appealing to the snake. Most reptile owners store frozen mice in their own freezers and heat them in warm water using a kitchen saucepan or microwave oven that is also used for food preparation (FDA 2017).

To the best of our knowledge, no mandatory measures have been established to assess the Salmonella status of rodents in supplier companies in Europe. This is attributed to snakes not being legally defined as pets, and therefore, feeder mice are not defined as pet food (EU regulation number 1774/2002). Recently, in the United Kingdom, imported commercial frozen mice have been associated with an outbreak of human salmonellosis originating from a large rodent breeding establishment in Europe (Kanagarajah et al. 2017). In this context, the objective of this study was to investigate Salmonella external and internal contamination of stored commercial frozen mice used to feed reptiles and obtained from the same supplier involved in the United Kingdom outbreak from 2016 (Kanagarajah et al. 2017).

Material and Methods

Commercial frozen feeder mice involved in the Salmonella Enteritidis human outbreak in United Kingdom (Kanagarajah et al. 2017) were supplied to the Animal and Plant Health Agency in Weybridge for testing at the time of the outbreak and stored at −20°C. The following different Feeder mice age categories were tested: pinkies (3–4 g), fuzzies (4.1–7 g), small (11–15 g), large (23–29 g), and extra-large (+30 g) mice. A total of 12 packs containing five individuals from each age group were tested, with the exception of the extra-large mice for which only 11 packs were available.

Results

Salmonella was isolated from 28.8% (n = 17) of the batches tested in one or more individuals within the batch. When batch types were compared, a significant difference was observed (Table 1). No large mice were positive for Salmonella spp. The most contaminated batch types were fuzzies (83.3%, n = 10), followed by small mice (41.7%, n = 5). Pinkies and extra-large mice showed a lower percentage of positive batches (8.3% for both, n = 1) (p < 0.05, Table 1).

When the location of the bacteria (externally or internally) was compared between positive batches, a significant difference was observed. Positive mice were mostly contaminated externally and less frequently internally (92.3% and 26.9%, respectively) (p < 0.05). Moreover, when the location of Salmonella was compared according to the type of mouse, a significant difference was observed. As reported in Table 1, fuzzies (28.3%) were more likely to be externally contaminated than pinkies, small, and extra-large mice (1.7%, 6.7%, and 3.6%, respectively) (p < 0.05). No statistically significant differences were found for Salmonella prevalence in viscera according to mouse type (Table 1).

The results obtained from individual internal organs of the fuzzies (n = 60) showed that three individuals from three different batches were internally colonized by Salmonella. In the first individual, liver, spleen, ceca, gut, bladder, and kidneys were all positive. Salmonella levels in liver, spleen, gut, and kidneys were 3 × 10⁵ CFU/g, bladder: 2 × 10⁵ CFU/g, and ceca: 4.9 × 10⁴ CFU/g. The second mouse was positive for Salmonella in ceca, intestines, and kidneys. Levels in intestines were 1.39 × 10⁵ CFU/g and kidneys: 4 × 10³ CFU/g. Cecal enumeration was negative. Finally, the third mouse was positive in the intestine only with a count of 2.5 × 10⁴ Salmonella CFU/g. All counts obtained from the exterior of the fuzzies were negative (n = 60).

Only Salmonella Enteritidis was isolated from the positive mice (n = 30); 53.3% of the phage typed isolates were Salmonella Enteritidis PT8 (n = 16), and 33.0% were Salmonella Enteritidis PT13 (n = 10). Moreover, four strains were classified as unknown phage type.

All the isolates were sensitive to the 16 antimicrobials tested.

Discussion

The results of our study demonstrate that all the frozen feeder mice categories tested, with the exception of the large mice, were contaminated by Salmonella. The contamination was especially prevalent on the external surface of the rodents, which could result in RAS human outbreaks associated with reptile food handling despite precautions taken when dealing with the reptiles themselves.

Epidemiological investigations have demonstrated several RAS human outbreaks (Bertrand et al. 2008, CDC 2003, 2012, 2014). Indistinguishable Salmonella Enteritidis and Salmonella Typhimurium strains shared among frozen feeder mice, pet snakes, and snake owners have been reported (Fuller et al. 2008, Lee et al. 2008, Cartwright et al. 2016, Kanagarajah et al. 2017). A recently published study demonstrated a relationship between strains from the commercial frozen mice and a recent Salmonella human outbreak in United Kingdom in which a significant number of people were infected over a prolonged time period (Kanagarajah et al. 2017). The commercial batches supplied were contaminated by a Salmonella Enteritidis PT8/PT13 strain, one of the most frequently involved serovar/phage type combination in human Salmonella outbreaks in Europe (EFSA and ECDC 2016). Batches of feeder rodents may become contaminated by Salmonella. However, to the best of our knowledge there is no legislation in Europe covering the risk of reptile food processing in relation to salmonellosis (Kanagarajah et al. 2017). Currently, feeder mice are usually sold frozen by suppliers and distributors (Lee et al. 2008). Before freezing, groups of mice are euthanized in a CO₂ chamber and during this process the bowels of the mice are likely to be evacuated. Exposure to the evacuated feces may further contribute to external contamination of the individuals. Moreover, it is known that freezing procedures do not kill Salmonella, and the organism is able to survive freezing for long periods (Dominguez and Schaffner 2009). Contamination could also be aggravated because distributors typically purchase frozen rodents in bulk and repackage them in smaller quantities before selling them to local stores (Cartwright et al. 2016). Thus, further handling increases the risk of cross-contamination between different batches and between individuals within a batch. Although the fuzzies qualitative analyses of the carcasses demonstrated a high prevalence of positive individuals, the results obtained in the quantitative study were negative. This may be due to the fact that the counts might be very low, and therefore, the detection through classical microbiology was not enough to be able to count. The fact that the mice carry the bacteria externally, although in low numbers, can lead to a future human outbreak, as has been seen in previous studies (Kanagarajah et al. 2017).

As this study demonstrated, frozen mice can also be contaminated internally; so even if mice were irradiated externally, internal contamination could persist and more powerful methods using gamma-irradiation would be needed. The source of contamination is likely to be in breeders and in the mouse processing environment. Rodent-to-rodent infection within a breeder rodent facility is common and is likely to be perpetuated across the generations of mice (Lee et al. 2008). This study demonstrated that the bacteria could be located in different internal organs, especially in intestines. Salmonella can survive for a long period in fecal pellets or dust and could be a source of rodent infection at the facility (Warwick et al. 2001, Lee et al. 2008).

To avoid Salmonella infections of reptile feed, different hygienic measures could be applied in mice production system. All-in-all out production, preventive hygienic measures (Carrique-Mas et al. 2009) feed, and drinking water acidification with organic acids and immune strategies based on passive and active immunity are the basic tools to reduce Salmonella (Vandeplas et al. 2010). In addition, modification of the diet by manipulating ingredients and nutrient composition with the intent of reducing animal susceptibility to Salmonella infection through prebiotic activity has also been implemented as a tool to help control Salmonella (Donadlson et al. 2008). Feed additives such as antimicrobial peptides, prebiotics, probiotics, and synbiotics that modify the intestinal microflora could be an aid to controlling enteric pathogens, but are unlikely to be successful in eliminating infection from a breeding mouse colony without other measures (Vandeplas et al. 2010). Other potential control methods such as the use of bacteriophages are also under study (Vandeplas et al. 2010). Individual packaging of mice before irradiation could help to avoid cross-contamination between mice. Moreover, the individual package should help minimize cross-contamination between mice and household facilities. Finally, improved statutory control of reptiles would be beneficial, but representative sampling would still present difficulties.

In conclusion, reptile feeder mice can carry Salmonella externally and internally; however, positive mice are mostly contaminated externally. Thus, lack of care in handling feeder mice could contribute to human Salmonella infections.