ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) is widely distributed in the environment and is recognized for its association with severe illnesses in both animals and humans. As effective treatments and preventive measures remain limited, it becomes crucial to understand the mechanisms underlying STEC-induced diseases. Animal models allow analyzing the pathophysiology of diseases. However, current models do not accurately reproduce the full spectrum of diseases caused by STEC. In this study, we discussed the key characteristics and constraints of five animal models (mouse, rabbit, chicken, dog, and pig) used to study STEC infections.
1. Introduction
Escherichia coli (E. coli) is a Gram-negative, motile, rod-shaped bacterium classified under the family Enterobacteriaceae (Do et al. Citation2020b). The majority of E. coli coexist as innocuous commensal symbionts in the gastrointestinal tract. (Do et al. Citation2019; Do et al. Citation2020b). Commensal E. coli strains predominantly inhabit the gastrointestinal tract and represent the major group of facultative anaerobes in the human colon (Do et al. Citation2019; Do et al. Citation2020b; Do et al. Citation2022). However, certain pathogenic E. coli strains have the potential to cause severe and life-threatening illnesses, which may lead to hospitalization and even death (Do et al. Citation2019; Do et al. Citation2020b; Do et al. Citation2022).
The pathotypes of E. coli are classified into five groups, which include enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, enteroaggregative E. coli, and Shiga toxin-producing E. coli (STEC; additionally known as enterohemorrhagic E. coli) (Do et al. Citation2020b). E. coli O157:H7 belongs to the group of STEC, which can colonize the gastrointestinal tract and lead to hemorrhagic colitis, characterized by bloody diarrhea (Tseng et al. Citation2015). STEC are recognized for their capacity to generate diverse variants of Stx1 (Shiga toxin type 1), Stx2 (Shiga toxin type 2), or both types of toxins. (Stratakos et al. Citation2018). Stx toxins are highly potent cytotoxins that can penetrate the epithelial barrier and disrupt protein synthesis in target endothelial cells (Jones et al. Citation2017; Stratakos et al. Citation2018).
STEC infections can have significant implications for human health, encompassing a range of clinical outcomes (Do et al. Citation2020b). The impact on human health is primarily associated with the production of Shiga toxins, which can lead to severe gastrointestinal symptoms, including diarrhea, abdominal cramps, and in some cases, bloody stools (Do et al. Citation2022). One of the prominent health concerns related to STEC infections is the development of hemolytic uremic syndrome, a condition characterized by the destruction of red blood cells, low platelet count, and kidney failure (Tseng et al. Citation2015).
The epidemiological background of STEC infections involves a diverse range of sources, including contaminated food products, waterborne transmission, and person-to-person contact (Do et al. Citation2020b). Consumption of undercooked ground beef, unpasteurized dairy products, and raw vegetables has been identified as common routes of transmission (Jones et al. Citation2017). Additionally, contact with animals and their environments, such as companion animals and/or industrial animals including pigs or cattle, can also contribute to the spread of STEC (Tseng et al. Citation2015).
Over the past four decades, since the initial identification of the E. coli O157:H7 outbreak, researchers have extensively studied STEC infections and Stx-mediated diseases using various animal models (Riley et al. Citation1983). The significance of animal infection models lies in their capacity to mimic the entire range of human infectious diseases, facilitating insights into the mechanisms involved in clinical disease, pathogen carriage, and transmission (Philipson et al. Citation2013). STEC pathogenesis involves oral infection followed by diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (Brilhante et al. Citation2019; Lee and Tesh Citation2019; Joseph et al. Citation2020). Animal models that can mimic STEC diseases are described below. A summary of animal models, treatments, and outcomes is presented in . This study describes the features and limitations of five animal models of STEC infections.
Table 1. Synopsis of Animal models, treatments, and results.
2. Pathogenesis
Stx-producing strains colonizing the intestine can cause diseases in mammals (Tarr et al. Citation2005; Jeong et al. Citation2018). Usually, STEC O157:H7 contains the locus of enterocyte effacement pathogenicity island, critical for bacterial attachment to the intestinal epithelium (Janke et al. Citation1989). The pathogenicity island harbours a type III secretion system and numerous secreted proteins crucial for attachment, including EspA, EspB, EspD, and Tir (Ha et al. Citation2008). Further, this island encodes intimin, an outer membrane adhesin that mediates STEC-host cell interaction (Malik et al. Citation2006). Some non-O157 STEC, such as O113, lack the locus of enterocyte effacement pathogenic island, and there is limited knowledge about the adhesion factor (Malik et al. Citation2006). Several researchers presumed that non-O157 STEC attach to epithelium via the putative adhesin Saa (Do et al. Citation2017; Stratakos et al. Citation2018; Do et al. Citation2022).
The pathophysiology of STEC is closely linked to its capacity to successfully colonize the mammalian intestine and produce Stxs. (Niewerth et al. Citation2001; Do et al. Citation2017). Stxs have been identified on the surface of polymorphonuclear leukocytes in the blood and feces of individuals affected by the infection (MacLeod et al. Citation1991). STEC has been found in the intestinal lumen and feces but does not cause bacteremia (MacLeod et al. Citation1991). The mechanisms responsible for Stx crossing the epithelial barrier remain unclear. Nevertheless, it is evident that the distribution of the Stx receptor globotriaosylceramide plays a crucial role in determining the site of tissue damage (Do et al. Citation2017). The presence of Gb3 in vascular endothelial cells and the host cell response to cellular damage collectively contribute to the pathology observed in Stx-associated diseases (MacLeod et al. Citation1991; Jeong et al. Citation2018). High Gb3 concentrations in the kidneys are associated with acute renal failure (Tarr et al. Citation2005; Lee and Tesh Citation2019). Gb3 has also been found to be present in the microvasculature of the colon and cerebellum, suggesting its possible involvement in Stx-related pathologies in these specific regions (Jeong et al. Citation2018; Lee and Tesh Citation2019).
3. Animal models of STEC infection
3.1. Mouse model
The pathogenesis of STEC infections was assessed in four mouse models: (1) mice orally treated with streptomycin, (2) mice intragastrically inoculated with streptomycin and mitomycin C- or ciprofloxacin, (3) intragastric inoculation, but not those treated with streptomycin, and (4) malnourished mice.
The results observed in infected mice can be influenced by several factors, such as the type of STEC strain used, the mouse strain, and the specific method of treatment and infection employed in the study. Most studies’ (at least, all studies described in this manuscript) methodology for the streptomycin-treated mice was adapted from previous studies by Myhal et al. (Myhal et al. Citation1982) and Wadolkowski et al. (Wadolkowski et al. Citation1990a), where adult CD-1 mice were administered streptomycin sulfate (5 g/L) in their drinking water one day before being orally infected with a streptomycin-resistant (Strr) STEC strain. In the oral infection procedure, the animals were exposed to the bacteria, which were suspended in a solution containing sucrose. They consumed the inoculum either by direct ingestion from a container in their enclosures or through the administration of the inoculum using a dropper. After infection, animals had ad libitum access to food and water containing 5 g/L streptomycin sulfate. The administration of streptomycin leads to a reduction of facultative anaerobes by more than 6 logs within 24 h, creating an environment that enables the Strr STEC strains to colonize the intestine at high levels (approximately 107 colony-forming units [CFUs]/g feces) for more than 1 week (Myhal et al. Citation1982). While the colonization of CD-1 and inbred strains of mice (e.g. DBA/2J) with O157:H7 is achievable using this approach, only certain E. coli O157:H7 strains (e.g. 86–24 Strr) exhibit virulence towards these animals while other E. coli O157:H7 strains (e.g. 933, 933cu) showed no virulence (no clinical signs). Moreover, successful infection in mice necessitates the use of a substantial inoculum (approximately 1010 bacteria). In streptomycin-treated mice, infection with an E. coli O157:H7 strain producing both Stx1 and Stx2 results in death primarily due to toxin-induced renal tubular necrosis (Wadolkowski et al. Citation1990b). According to the results of A.R. Melton-Celsa et al. (Melton-Celsa et al. Citation1996), when these mice were fed a STEC O91:H21 strain that produces the Stx2d variant, known to be activated by elastase present in the intestinal mucus, the oral 50% lethal dose was determined to be less than 10 bacterial cells. And this is accordance with the report of J.F. Kokai-Kun et al. (Kokai-Kun et al. Citation2000). Mice infected with this highly pathogenic strain succumb to renal tubular necrosis as a fatal outcome. As a result, streptomycin-treated mice are not a suitable model for simulating STEC-mediated diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. However, they can be effectively utilized to evaluate the virulence of STEC after oral challenge, the induction of Stx-mediated renal tubular necrosis, and the efficacy of Stx inhibitors. We previously found that an Stx2 antibody protected streptomycin-treated STEC-infected mice (Wadolkowski et al. Citation1990b; Lindgren et al. Citation1993).
Two separate experiments employed a modified version of this mouse model, where phages were administered either during or after intragastric infection with O157 STEC strain. (Fujii et al. Citation1994; Zhang et al. Citation2000). One experiment administered mitomycin C to streptomycin-treated mice during intragastric inoculation with the O157:H- strain E32511/HSC (Fujii et al. Citation1994). Mice exposed to streptomycin and mitomycin C treatment, along with a high bacterial load (>109 CFUs per mouse), experienced mortality and acute encephalopathy. In the subsequent trial, CD-1 mice treated with streptomycin were given ciprofloxacin at dosages leading to a 1000-fold decrease in the CFUs of the infecting strain excreted in the feces (Zhang et al. Citation2000). Ciprofloxacin triggers the activation of the phages that carry the gene for Stx2, consequently leading to an elevation in the level of Stx2 production by the STEC strain 1:361R in both in vitro and in vivo conditions (Zhang et al. Citation2000).
Approximately two-thirds of the experimental mice that received ciprofloxacin treatment and were infected with 1:361R died, while the untreated mice infected with 1:361R alone survived. The discovery that ciprofloxacin induced mortality in streptomycin-treated 1:361R-infected mice, and that death correlated with elevated levels of free fecal Stx, implies that this model could potentially render a mouse-attenuated STEC strain virulent (Zhang et al. Citation2000).
In the third model, C3H/HeN mice (lipopolysaccharide [LPS]-responders) or C3H/HeJ mice (LPS-nonresponders) were intragastrically inoculated with 107–108 CFUs of the Stx2-producing O157:H7 strain 86–24 or its derivative, 86BL (Karpman et al. Citation1997). As a control group, Karpman et al. utilized strain 87-23, an O157:H7 strain that does not produce Stx, and was isolated during the same outbreak as strain 86–24 (Greene et al. Citation1988). C3H/HeN mice exhibited more severe disease, suggesting that the response to LPS played a important part in the development of STEC inducing disease. Furthermore, only mice infected with an Stx2-producing strain exhibited the mesangial matrix expansion in renal glomeruli. Nonetheless, mice infected with the toxin-negative O157:H7 strain also exhibited signs of O157-mediated pathology, including loose stools and vascular congestion. Furthermore, both strains were found to invade the bloodstream of mice. On the other hand, the non-O157 control strain elicited only mild systemic symptoms, lasting for less than one day.
As part of the fourth model, malnutrition was induced in female C57BL/6 mice by subjecting them to a 5% protein diet for 2 weeks before and after intragastric infection with the O157:H7 strain, N-9 (Kurioka et al. Citation1998). Malnourished mice, in contrast to the control mice fed a 25% protein diet, experienced fatal outcomes when exposed to an inoculum surpassing 106 CFUs. Malnourished mice developed systemic and neurological signs and died, as observed in other mouse models. Moreover, malnourished mice had no appreciable kidney damage but developed cerebral hemorrhage, which could be the cause of death. The absence of kidney damage observed in malnourished mice, unlike streptomycin-treated mice, could potentially be attributed to the nutritional status of the animals.
Although none of the mouse models fully reflects the pathogenesis of STEC in humans, certain systems show promise as models for the most severe aspects of STEC infection, particularly the hemolytic uremic syndrome. However, mouse models are more accessible than larger animal models (such as rabbit, chicken, dog, and pig). Also, another advantageous aspect of the mouse models is their ability to effectively showcase Stx-mediated damage. Hence, they prove valuable for assessing the effectiveness of therapies targeting the toxin.
3.2. Rabbit models
The rabbit model for STEC infection was initially described by J.J. Farmer et al, where 5- to 10-day-old rabbits were intragastrically fed with E. coli O157:H7 (108 CFU/rabbit), contributing to the development of gastroenteritis in these animals (Farmer et al. Citation1983). Diarrhea was not observed in older rabbits, guinea pigs, mice, or young rhesus monkeys infected with O157. Pai et al. specifically reported the infection of young rabbits with strain O157:H7 (Pai et al. Citation1986). Rabbits exposed to the highest inoculum experienced severe diarrhea and mortality. Rabbit diarrheagenic E. coli (RDEC) is a subset of STEC, and RDEC-1 is a prototype strain recognized for inducing diarrhea in weaned rabbits. R. Sjogren et al. utilized nalidixic acid-resistant variant RDEC with an Stx1-converting phage (Sjogren et al. Citation1994). The strain RDEC-H19A was employed to investigate the impact of Stx1 in enteritis-induced infections, and infection with 108 CFU of RDEC-H19A induced diarrhea more rapidly and with greater frequency compared to the RDEC control strain (Sjogren et al. Citation1994). Additionally, the cecum and proximal colon of RDEC-H19A-infected rabbits exhibit subserosal hemorrhage and submucosal edema on day 7 post-inoculation (Sjogren et al. Citation1994). Rabbits infected with the Stx1-producing strain exhibited vascular changes in the submucosal venules, as observed in the study (Sherman et al. Citation1988). A study intragastrically fed different STEC serotypes of 108 E. coli cells (including O157, O26, O111, O113, O121, and O145) to weaned rabbits and found that most strains attached intimately to ilea, ceca, and colons, and induced diarrhea (Sherman et al. Citation1988). Moreover, the tested strains strongly attached to the intestinal epithelium.
Young rabbits intragastrically administered sodium bicarbonate and a Stx1-producing O157:H7 strain died of diarrhea (Pai et al. Citation1986). The histological analysis of the colons of rabbits given Stx preparations showed mucosal damage. Moreover, purified Stx1 (0.2 μg/kg) caused diarrhea in 50% of treated animals (Richardson et al. Citation1992). A few animals showed occult blood in their stools, but overt bloody stools were not observed. Several animals developed central nervous system (CNS) symptoms, such as paresis and intestinal edema, and approximately 50% of the animals exhibited vascular changes in the intestinal submucosa. In a particular study, rabbits injected intravenously with a substantial bolus of Stx2 (2 μg/kg) displayed symptoms such as hemorrhagic diarrhea, flaccid paresis, convulsions, and CNS involvement (Fujii et al. Citation1996). In another study, it was observed that the continuous infusion of Stx2 into the peritoneum resulted in diarrhea and intestinal lesions that were similar to those seen in cases of hemorrhagic colitis (Keenan et al. Citation1986; Barrett et al. Citation1989). When purified Stx or Stx1 was injected into ileal loops, it led to fluid accumulation and the development of microscopic lesions. In addition, most villous cells were shed or underwent apoptosis 24 h post-inoculation.
Rabbit models are useful for the study of mechanism of attaching and effacing lesion. and Stx. Also, the outcomes of models are similar to its of pig models which are highly expensive. However, RDEC had species speficity in in vivo infectivity, and not precisely mimic the course of STEC infection in humans. Also, the availability and cost of using rabbit models can be prohibitive for some researchers.
3.3. Chicken models
Two studies orally inoculated chickens with E. coli O157:H7 and observed that this STEC strain colonized the cecum and colon and was shed in the feces for more than 5 months (Beery et al. Citation1985; Schoeni and Doyle Citation1994). Another study found attaching and effacing lesions in two of seven chicks fed the O157:H7 strain ATCC 43889 (Sueyoshi and Nakazawa Citation1994).
Chicken models serve as valuable tools for exploring STEC colonization and understanding the mechanisms behind intestinal damage caused by attaching and effacing lesions. However, the chicken model does not accurately replicate the pathogenesis of STEC in humans and is less accessible, posing a challenge for researchers in terms of availability.
3.4. Dog models
The use of greyhound dogs as a model for studying STEC infection is still in its early stages of development. In this model, the symptoms of STEC disease closely resemble those observed in idiopathic cutaneous and renal glomerular vasculopathy (CRGV) (Proulx et al. Citation2001). However, unlike STEC-associated illnesses, CRVG is known for causing skin lesions on the abdominal area and hind legs (Proulx et al. Citation2001). In dogs, CRGV is also characterized by symptoms such as thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure (glomerular necrosis, capillary endothelial damage, fibrin accumulation, and endothelial cell hypertrophy and proliferation) (Proulx et al. Citation2001). STEC is suspected to cause CRVG. Even though animals with CRVG do not exhibit diarrhea, the clinical presentation bears resemblance to that of hemolytic uremic syndrome. Therefore, dog models may elucidate the process of hemolytic uremic syndrome and foster the development of effective therapies for this syndrome.
Dog models exhibit clinical features akin to hemolytic uremic syndrome, a critical aspect for human research. However, their utilization is associated with high costs and limited accessibility, posing challenges for many researchers.
3.5. Pig models
E. coli is known to cause various diseases in pigs (Gunzer et al. Citation2003; Sato et al. Citation2017). However, it is important to note that these diseases are often associated with other risk factors (Gunzer et al. Citation2003; Sato et al. Citation2017). For instance, in cases of E. coli-induced neonatal diarrhea in pigs, the health condition of the sow, the quality of colostrum provided, and the piglets’ viability after birth are crucial factors that significantly influence the disease's development (Renzhammer et al. Citation2020). STEC, especially producing Stx2e cause edema, increasing morbidity and mortality in weaned piglets (Do et al. Citation2017). The Shiga-toxin produced by Stx2e-encoding STEC exhibit weak enterotoxigenic properties, however, they showed high cytotoxicity to porcine vascular endothelial cells (MacLeod et al. Citation1991). In the early stage of the disease, viable counts of E. coli (109 CFU/g) are commonly detected in the small intestine of pigs (Do et al. Citation2020a; Do et al. Citation2020b; Do et al. Citation2021).
Gnotobiotic piglets orally infected with O157 STEC strains exhibit intestinal and extra-intestinal symptoms, including watery diarrhea 3–4 days after challenge. The severity of mucosal damage is linked to the presence of attaching and effacing lesions in colonocytes (Gunzer et al. Citation2003). Up to 90% of animals develop neurological symptoms 2–3 days after infection, including ataxia, lateral recumbency, and death (Gunzer et al. Citation2003). The disease presents histologically as small hemorrhagic foci and necrosis in the cerebellum (Gunzer et al. Citation2003). Furthermore, this animal model allows for oral inoculation without the need for antibiotic pretreatment, making it convenient for research purposes. It provides a valuable platform to study the mechanisms of pathogenicity in mutant STEC strains and explore gene regulation in both bacteria and host organisms (Gunzer et al. Citation2003).
The pig model is a useful means for investigating the effects of STEC pathogenicity factors with the application of mutant strains. Furthermore, it functions as a valuable tool for evaluating gene regulation in both the bacteria and the host. Nevertheless, for a more comprehensive understanding of the mechanisms underlying human STEC disease and the development of intervention strategies informed by animal data, it is imperative to thoroughly characterize the model in terms of how closely it mirrors hemolytic uremic syndrome in humans.
4. Conclusions
Animal models do not accurately mimic the full clinical spectrum of diseases caused by STEC. Selecting appropriate animal models is crucial and should be based on the specific scientific inquiry at hand. For research aimed at gaining insights into the biology of an organism, model systems that closely mimic the natural conditions of the intestine should be employed. Such models offer a more accurate representation of the biological processes and interactions that occur in the intestinal environment. Generally, we recommend mouse models or rabbit models for investigating Stx-mediated damage because of low-cost and readily available to all researchers. In the pursuit of devising control measures for Shiga toxin-producing Escherichia coli (STEC) in pigs, given its substantial economic impact on the swine industry, it is advisable to employ available pig models, as they are most relevant for studying the majority of STEC strains. For researchers seeking to investigate disease forms closely resembling human conditions, the utilization of a dog model is recommended, as it manifests symptoms highly analogous to hemolytic uremic syndrome.
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No potential conflict of interest was reported by the author(s).
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