Bacterial species to be considered in quality assurance of mice and rats

Abstract

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Bacteria are relevant in rodent quality assurance programmes if (a) the animals are at risk and (b) presence in the animals makes a difference for animal research or welfare, for example because the agent regulates clinical disease progression or impacts its host in other ways. Furthermore, zoonoses are relevant. Some bacterial species internationally recommended for the health monitoring of rats and mice, that is, Citrobacter rodentium, Corynebacterium kutscheri, Salmonella spp. and Streptococcus pneumonia, are no longer found in either laboratory or pet shop rats or mice, while there is still a real risk of impact on animal research and welfare from Filobacterium rodentium, Clostridium piliforme, Mycoplasma spp., Helicobacter spp. and Rodentibacter spp., while Streptobacillus moniliformis may be considered a serious zoonotic agent in spite of a very low risk. Modern molecular techniques have revealed that there may, depending on the research type, be equally good reasons for knowing the colony status of some commensal bacteria that are essential for the induction of specific rodent models, such as Alistipes spp., Akkermansia muciniphila, Bifidobacterium spp., Bacteroides fragilis, Bacteroides vulgatus, Faecalibacterium prausnitzii, Prevotella copri and segmented filamentous bacteria. In future, research groups should therefore consider the presence or absence of a short list of defined bacterial species relevant for their models. This list can be tested by cost-effective sequencing or even a simple multiple polymerase chain reaction approach, which is likely to be cost-neutral compared to more traditional screening methods.

Keywords

disease model ethics and welfare microorganism organisms and models quality assurance/control

Introduction

Mice and rats are quality assured by screening for the absence of specific pathogens, for example as recommended in guidelines issued by the Federation of European Laboratory Animal Associations (FELASA).¹ However, many of those bacteria screened for are seldom found in barrier-protected rodents,² while the access to modern molecular techniques has revealed other and often non-cultivable bacteria as serious modulators of animal disease models. It has therefore been proposed that symbiotic bacteria with an impact on rodent models are also made part of routine quality assurance,³ and furthermore, it has been discussed whether some animal models, for example within immunology, would actually be improved if the immune system had been challenged with a more diverse microbiota, even if this included some pathogens.⁴

The FELASA pathogens

The main objective of the most recent FELASA health-monitoring recommendations is ‘to harmonize health monitoring programmes, which will help to improve knowledge about the microbiological quality of animals used in research’.¹ These recommendations define the term ‘microbiological quality’ as ‘the occurrence of infectious agents’.⁵ The agents listed by FELASA are shown in Table 1.

Table 1.

Bacterial agents recommended by FELASA for routine screening in breeding colonies of rats and mice¹ and the prevalence with which they have been diagnosed globally in recent surveys from laboratory animal colonies, pet shops and single cases.

	Screening recommended by FELASA
	Mice	Rats	Global prevalence in laboratory rats and mice^2,16–19	Global prevalence in pet shop rats and mice^8,20–22	Zoonotic
Filobacterium rodentium (Cilia-associated respiratory bacillus)	None	Annually	(+)	0	–
Citrobacter rodentium	Annually	None	0	0	–
Clostridium piliforme	Annually	Every 3 months	++	+	(†)
Corynebacterium kutscheri	Annually	None	0	0	–
Helicobacter spp.	Every 3 months	Every 3 months	+	+++
Mycoplasma spp.	Annually	Every 3 months	+	+	–
Rodentibacter spp. (Pasteurella pneumotropica)	Every 3 months	Every 3 months	+	+	†
Salmonella	Annually	Every 3 months	0	0	†
Streptobacillus moniliformis	Annually	Annually	0	+	†
β-Hemolytic streptococci	Every 3 months	Every 3 months	+	0	†
Streptococcus pneumoniae	Every 3 months	Every 3 months	0	0	†

ND No data; 0 Not found; (+) Prevalence < 5%; + Prevalence = 5–33%; + + Prevalence = 33–66%; + + + Prevalence > 66%; - Not zoonotic; (†) Single cases reported; † Several cases reported.

However, it is questionable whether the bacteria currently listed represent the major risk factor for the unsuccessful use of rodents as models for human disease, since some of these, i.e. Citrobacter rodentium, Corynebacterium kutscheri and Streptococus pneumonia, have not been found in any microbiological surveys published within the last 15 years. This is also the case for Streptobacillus moniliformis and Salmonella spp., but S. moniliformis was isolated from barrier-bred laboratory mice back in 1990¹⁴ and from pet rats¹³ and human patients^15,16 as late as 2017, and Salmonella spp. is present with a low prevalence among wild rodents^17,18 and is one of the most common food-borne infections.¹⁹ The majority of those agents found cause mostly latent infections, but their routine screening can be argued on their ability to interfere with various models even in the absence of clinical manifestations. Rodentibacter spp. (previously known as Pasteurella pneumotropica but now reclassified as eight different species²⁰), while probably unable to cause pneumonia in immune-competent mice,^21,22 may be isolated from abscesses,^23,24 may alter cytokine expressions,²⁵ and may have some additional impact on immune-deficient mice.^22,26 A few of the numerously described Helicobacter spp. may cause clinical disease.^27–32 Clostridium piliforme may cause Tyzzer's disease,^33–36 and although infections with it are often latent due to genetic traits in rats³⁷ and mice,³⁸ research interference may still be expected due to dysfunction of the liver.³⁹ Mycoplasma pulmonis and M. arthritidis may cause respiratory, eye and joint-related conditions and may affect reproduction, at least if enhanced by other factors.^21,40–45 However, even in the absence of clinical symptoms, it may have impact on certain models.^46–50 Filobacterium rodentium (formerly known as cilia-associated respiratory (CAR) bacillus⁵¹), while usually asymptomatic,⁵² is the cause of chronic respiratory disease in rats,^53,54 and BALB/c mice.^54,55 There is only one reported case that infection with group B β-haemolytic streptococci, S. agalactiae, has caused general abscesses and mortality,⁵⁶ and, therefore, this is an example of an agent that does not necessarily require routine monitoring. Rodentibacter spp.,⁵⁷ Helicobacter spp.,⁵⁸ C. piliforme,⁵⁹ M. pulmonis,⁶⁰ and F. rodentium⁶¹ can be diagnosed by polymerase chain reaction (PCR), and although serology may be considered less sensitive and specific it is also commonly applied for C. piliforme,³⁵ M. pulmonis⁶² and F. rodentium.⁶³ β-haemolytic streptococci need cultivation on a blood agar prior to serotyping or PCR of β-haemolytic colonies.⁶⁴

Other facultative pathogens

Various other facultative pathogens are common agents in rodent facilities (Table 2). Staphylococcus spp. may have more or less the same impact as Rodentibacter spp.^65,66 Bordetella hinzii, and B. pseudohinzii isolated from the respiratory organs of laboratory rats and mice^67,68 are known to cause mild respiratory symptoms and disturb murine models of pulmonary disease.^69,70 Pseudomonas aeruginosa may cause conjunctivitis, rhinitis and pneumonia in immune-deficient or stressed animals⁷¹ as well as in transgenic mouse models of cystic fibrosis.⁷² Klebsiella oxytoca has been associated with otitis media, urogenital tract infections and pneumonia in mice.^73,74 Nude mice may develop a syndrome characterized by scaling and crusty skin, the most common agent of which is Corynebacterium bovis,^75,76 but severe disease is probably multifactorial.⁷⁷ Other Corynebacterium spp., such as C. renale⁷⁸ and C. mastitidis,⁷⁹ may cause urogenital suppuration in rats and mice. All of these bacteria can be diagnosed by PCR.^69,80–84

Table 2.

Some specific facultatively pathogenic bacteria in rats and mice.^66–80

	Phylum	Disease
Bordetella hinzii	Proteobacteria	Respiratory
Bordetella psedohinzii	Proteobacteria	Respiratory
Corynebacterium spp.	Actinobacteria	Skin, urogenital
Klebsiella oxytoca	Proteobacteria	Ears, respiratory, urogenital
Pseudomonas aeruginosa	Proteobacteria	Eyes, respiratory
Staphylococcus spp.	Firmicutes	Skin, respiratory

Gut commensals with an impact on mouse and rat models

The access to molecular techniques, such as PCR, 16S rRNA gene sequencing and metagenomic sequencing,⁸⁵ for a deeper characterization of the rodent microbiota has revealed that many of those bacteria, which have been difficult to isolate and identify previously have a profound impact on a range of animal models.⁸⁶ Much of this impact is complex and is most likely driven by several bacteria in conjunction. However, enough information is available in order to identify a number of specific bacteria about which information on their status will be important for a range of research groups (Table 3). All of these, that is, Alistipes spp.,⁸⁷ A. muciniphila,⁸⁸ bifidobacteria,⁸⁹ Bacteroides spp.,⁹⁰ F. prausnitzii,⁹¹ Prevotella spp.⁹² and segmented filamentous bacteria (SFBs),⁹³ can be detected by PCR.

Table 3.

Some specific commensal gut bacteria with a documented impact on animal models.^90–135

	Phylum	Anti-inflammatory (A) Pro-inflammatory (P) (+)
Alistipes spp.	Bacteroidetes	P
Akkermansia muciniphila	Verrucomicrobia	A
Bifidobacterium spp.	Actinobacteria	A
Bacteroides fragilis	Bacteroidetes	P
Bacteroides vulgatus	Bacteroidetes	P
Faecalibacterium prausnitzii	Firmicutes	A
Prevotella copri	Bacteroidetes	P
Proteus mirabilis	Proteobacteria	P
Segmented filamentous bacteria (Candidatus savagella)	Firmicutes	P

Alistipes

Alistipes spp. appear to impact neuropsychiatric mouse models. Increased abundances have been correlated with a low level of serotonin in the ileum in autistic mice⁹⁴ and the expression of depressive behaviour in grid floor housed mice.⁹⁵

Akkermansia

Akkermansia muciniphila is the only well-described member of the genus, and the only one observed in rodents to date.⁹⁶ It feeds on mucin in the gut.⁹⁷ In both humans and mice, if present in the colony, it constitutes 0.1–3% of the total gut microbiota, but it may only be present in young mice.⁸⁸ Its anti-inflammatory potential is linked to the reduced incidence or severity of disease, as in murine models for type 1 diabetes^98,99 and diet-induced obesity,¹⁰⁰ while it seems to have a co-inducing impact on the development of azoxymethane-induced colon tumours in mice.¹⁰¹ It is a potential target of dietary intervention studies,¹⁰² for example feeding with the popular prebiotic xylooligosaccharide has been found to propagate A. muciniphila in mice.¹⁰³ Therefore, it can be problematic to use mice from colonies free of A. muciniphila in studies of prebiotics.⁹⁸

Bifidobacterium

Bifidobacterium spp. are anaerobic and frequently branched bacteria, and are major members of the colon microbiota of mammals.¹⁰⁴ High abundances of bifidobacteria are strongly correlated to low levels of inflammation in mice⁸⁹ and leptin in rats.¹⁰⁵ They are among the most popular probiotics for humans, and mice are used for the development of them.^106–108 Prebiotics tested in mice often act directly on the abundance of Bifidobacterium spp,^89,109,110 and it is therefore crucial that mice in such studies carry this organism.¹⁰⁴

Bacteroides

B. fragilis is normally considered a gut commensal, but toxin-producing strains may be regarded as pathogenic, for example they may induce colonic tumours in multiple intestinal neoplasia mice,¹¹¹ and feeding gnotobiotic mice B. fragilis toxins causes symptoms of diarrhoea.¹¹² In contrast to this, the polysaccharide A (PSA) from B. fragilis protects against Helicobacter hepaticus-induced colitis in mice; most likely because it prevents IL-17 secretion.¹¹³ Through its stimulation of the dendritic cells, PSA is important for gut immune maturation and the balance between T helper cells type 1 and 2, as well as for the gut response to pathogens.¹¹⁴ B. fragilis is also known to protect against intestinal natural killer T cell-mediated oxazolone-induced colitis in mice.¹¹⁵ Feeding B. fragilis to the maternal immune activation mouse model of autism reduces symptoms, which may be due to the normalization of one specific gut metabolite, 4-ethylphenylsulfate.¹¹⁶ B. vulgatus seems to enhance IBD in HLA-B27 transgenic rats¹¹⁷ and IL-10 knockout mice,¹¹⁸ and it increases in abundance in old mice.¹¹⁹

Faecalibacterium

Faecalibacterium prausnitzii is a clostridium-related bacterium that is among the most abundant members of the human gut microbiota, where it is an important producer of butyrate and appears to play an important role in the breakdown of complex carbohydrates.^120,121 In mice, it reduces the severity of various models of IBD, and it may be a key target in IBD intervention studies.^122–124 It is found spontaneously in some mouse colonies,¹²⁵ while it is absent in others.¹²⁶ In addition, other but less well-defined Clostridium spp. are known to induce colonic regulatory T cells.¹¹⁵

Prevotella

Gram-negative mostly anaerobic Prevotella spp. are observed in the gastrointestinal tract, including the oral cavity. P. copri may increase the severity of dextran sodium sulfate (DSS)-induced colitis in mice.¹²⁷ The lower response to DSS induction observed in Caspase-3 knockout mice may be counteracted by cohousing with wild type mice, which significantly increases the abundance of Prevotella spp. in the knockout mice.¹²⁸ Tumour-bearing mice with azoxymethane/DSS-induced colorectal cancer may have decreased abundances of Prevotellaceae,¹⁰¹ while a high abundance of Prevotellaceae in the gut of leptin-deficient obese mice correlates to an impaired glucose tolerance.⁸⁸

Proteus

Proteus mirabilis is a common inhabitant of the murine gut. So far, it has been considered a commensal; however, it seems to increase in abundance in mouse models of Parkinson's disease, and the transfer of P. mirabilis isolated from such models to other mice damages dopaminergic neurons and motor functions.¹²⁹

SFBs (Candidatus Savagella)

SFB is a more popular term for the Clostridium cluster I-related Candidatus Savagella. They are sometimes wrongly referred to as Candidatus Arthromitus, but these are Lachnospiraceae-related bacteria found in arthropods and not in mammals.¹³⁰ Species found in mice and rats are closely related but different from one another.¹³¹ Mouse colonies differ in terms of SFB status,^93,132 which is crucial to know, as SFBs strongly influence immune functions such as IgA production, T-cell response and intestinal T helper cell type 17 (T_h17) induction.^132–135 The recolonization of germ-free mice with SFBs induces a full reconstitution of all CD4-positive T cells¹³⁶ and increases the expression of the major histocompatibility complex (MHC) class II.¹³⁴ Since T_h17 is important in models of Crohn's disease, an impact of SFBs on IBD models should be expected, for example reconstituting the SCID adoptive transfer model with a SFB-containing microbiota enhances intestinal inflammation.¹³⁷ SFBs also promote arthritis development in K/BxN mice¹³⁸ and enhance experimental allergic encephalomyelitis development in myelin oligodendrocyte glycoprotein-injected mice,¹¹⁵ while they reduce the incidence of type 1 diabetes in female NOD mice.¹³⁹

Discussion

To select specific bacteria for consideration before using animals for a specific study, it seems relevant to consider agents if (a) the animals are in reality at risk and (b) if infections in the animals will make a difference for animal research or welfare, for example because the agent regulates clinical disease progression or in other ways has an impact on animal models. To this should be added that it is relevant to consider a zoonotic potential. This also implies that if neither (a) or (b) are fulfilled, then there is no need for specific screening. C. rodentium, C. kutscheri, Salmonella spp. and S. pneumonia are, in reality, absent in laboratory rats and mice. Furthermore, a single reported case on β-haemolytic streptococcal disease⁵⁶ is questionable as a justification for frequent global screening in favour of some of the other agents presented here. All of the bacteria listed in Tables 2 and 3 fulfil requirements (a) and (b), but they are obviously not equally relevant for all types of research and, therefore, it may be up to the individual facilities and the needs of their research groups to decide which ones should be routinely screened for. However, it is no longer sufficient only to secure the sole absence of pathogens. Some bacteria are crucial for some models but ruin other models, for example SFBs facilitate IBD¹³² but counteract type 1 diabetes,¹³⁹ F. prausnitzii and A. muciniphila reduce severity in IBD¹²⁴ and type 1 diabetes models,⁹⁸ respectively, but this is precisely why they are targets for intervention.^99,123 F. rodentium, C. piliforme, Mycoplasma spp., Helicobacter spp. and Rodentibacter spp. also fulfil requirements (a) and (b). In addition, it may be argued that F. rodentium, C. piliforme, and Mycoplasma spp. represent organisms for which there is a real risk of outbreak of clinical disease with a high prevalence in a colony. It may also be argued that S. aureus is as important as Rodentibacter spp., since reports about abscesses are more or less similar.¹⁴⁰ However, laboratory rodents probably catch this agent from their human caretakers, as it is extremely rare in wild rodents,¹⁴¹ and, therefore, it is far more difficult to keep out of barrier facilities. Zoonotic agents, such as S. moniliformis, despite presenting a low risk, may be included; however, it could be argued that Leptospira spp., also latently present in the wild rodent population,^142,143 are equally important as a zoonosis, and it is unlikely to isolate any of these two agents in modern animal facilities.

Sequencing costs are rapidly declining, and the fast, cost-effective screening of samples for unknown species using portable, real-time sequencers will soon change molecular diagnostics.^103,144 Even if sequencing is not an option, most bacteria can still be monitored simply by using PCR. Since it is likely that the list of bacteria with a research impact will continue to grow, it may well be best to reduce the number of organisms routinely screened for at breeders and to allow local needs to direct what should be included in further testing. On the other hand, with the use of multiplex PCRs it may be possible to screen for a longer list of bacteria, routinely or on request, which may be seen as a competitive move for a commercial breeder. As positive results for wanted organisms need less frequent re-confirmation and as laborious cultivations are no longer required, which also eliminates the need for transporting live animals to laboratories, the costs of bacteriology will be reduced dramatically.

In conclusion, research groups may benefit from having a short list of the presence or absence of defined and relevant bacterial species for the animals they use. The list will probably need current revision, as new bacteria with an impact will be involved, and as increase in knowledge and technology will lead to a higher resolution in identification. Routine screening can be performed using cost-effective sequencing or simple multiple PCR approaches. With fewer pathogens on the list and with the transition from cultivation to molecular methods, this is likely to be cost-neutral.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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