Helicobacter hepaticus and H. bilis have become a major concern for many animal care and use programs. In 1994, Taconic began testing its Barrier Units for Helicobacter organisms by utilizing accepted culture techniques. In 1997, Taconic's contract testing laboratory began utilizing PCR technology for detection of Helicobacter organisms. Since 1997, all of Taconic's Barrier Units have been tested quarterly for Helicobacter sp. by PCR. Gnotobiotic isolators (MIG's) are tested semiannually. Taconic presently uses a PCR assay that detects rodent Helicobacter sp. as well as PCR assays for the detection of H. bilis, H. hepaticus, and H. pullorum.
H. hepaticus and H. bilis (both urease +), which are associated with pathology in mice and rats, have not been found in any Taconic produced mice or rats utilizing culture, histopathology and/or PCR techniques. In 1998, H. rodentium (urease negative) was detected in Taconic's IBU 16 and Surgery barrier. During 1999 Taconic began to eradicate H. rodentium from these two barriers due to its questionable status, even though no signs of clinical disease were apparent in stressed animals. Since 2002 Taconic's commercial animals have been totally free of all known rodent Helicobacter species.
In 1999, Dr. Steven Weisbroth of Anmed/Biosafe prepared the attached paper, "The Rodent Helicobacters: Present Status". This paper was written to clarify the questions most frequently asked by clients concerning the current knowledge of rodent Helicobacters. Starting on page 8, guidelines relative to rodent Helicobacters are proposed for Facility Managers regarding the procurement and maintenance of rats and mice. We hope you will find Dr. Weisbroth's paper a useful reference for decision making regarding how to manage the Helicobacter question in your animal program.
For questions or additional information, contact your Taconic Area Technical Representative.
The index cases of Helicobacter hepaticus hepatitis were first reported as incidental histopathologic findings from NCI mouse colonies at the Frederick (MD) Cancer Research Center (FCRC) in the Fall of 1992 (1). Through an intensive diagnostic and research effort, it was learned that the condition was caused by a newly recognized helical bacterium, soon to be designated Helicobacter hepaticus, isolated from affected livers. The agent could be passed to satisfy Koch's postulates as an incitant of the hepatitis, and as was later recognized , of the hepatomas induced as late stage sequelae in certain mouse strains (2). These cases, and the reports about them making their way into the literature served as a wake up call to the laboratory animal science community that a new chapter was being opened in the annals of rodent disease.
Indeed, in the journals and platform sessions of regional and national meetings in the 5 years since that time one could not find a topic in laboratory animal science that has generated scrutiny as intensive as that accorded the rodent helicobacters. For example, in a recent review of the subject, over 260 reference citations were given, an average of 40-50 /year (3). The good news is that shape and dimensions can be put on the subject of rodent helicobacteriosis. This information can be applied to such issues as methods of detection, significance of infection with the various Helicobacter species, likely clinical manifestations in various host stocks and strains, institutional concerns with standards for procurement and methodology for surveillance and eradication.
From the experience with H. hepaticus and enterohepatic disease at the FCRC it was apparent very early that even with similar exposure modes, males were more affected than females, that certain host strains, e.g. scid, A/J, C3H/HeN, BALB/cAn, DBA/2 N, CBA/J and all other immunodeficient strains were affected more than others, while certain other stocks and strains, e.g. C57BL/6N, B6C3F1, B6D2F and CD2F1, appeared to be colonized, but otherwise nonlesioned and resistant enteric carriers.
Since that time, the general experience has been as follows. In enzootically infected colonies of susceptible strains, the condition appears to be contracted early in life (most probably, but not exclusively, from carrier dams). Liver lesions of focal necrosis and focal nonsuppurative inflammation initially develop in mice 1-4 months of age (experimentally, about 4 weeks after orogastric lavage) but progress to include hepatocytomegaly, bile duct hyperplasia and cholangitis by 6-8 months of age. In at least one susceptible strain (A/JCr), the liver lesions progressed to hepatocellular carcinoma (2). Spiroform bacteria typical of Helicobacter in such lesions, are demonstrable by silver stains (e.g. the Steiner modification of the Warthin-Starry stain), especially in bile canaliculi. The term "chronic active hepatitis" was applied to these infections to denote the persistent infection, active necrosis of hepatocytes and hepatic parenchymal loss and repair that progress throughout the insidious course (1).
Other aspects of the clinical syndrome have been recognized, as well, particularly typhlocolitis with proctitis and rectal prolapse in immunodeficient mice infected with H. hepaticus (4) and typhlocolitis with hepatitis in immunodeficient mice infected with H. bilis (5, 6). Moreover, H. bilis has been diagnosed as a cause of typhlocolitis in athymic rats (7). However, the specific significance of Helicobacter infection of the colon and rectum in the pathogenesis of rectal prolapse is uncertain. On the one hand, rectal prolapse has been associated with helicobacteriosis in a number of immunodeficient strains (4, 6). On the other, especially in transgenic knockout mice with perturbation of the immune system (e.g., IL-2 and IL-10 deficient, TCR Ñ , Ò ,× positive, MHC-II and Ga-i2 deficient), rectal prolapse with colitis has been observed in the absence of (diagnosed) helicobacteriosis (8, 9, 10).
The immune response to infection is prompt and appears proportional to the intensity of infection or degree of tissue invasion, but may not confer protection (3). Unlesioned and naturally resistant mouse strains may be colonized by H. hepaticus without development of a detectable antibody response (4). Both whole cell sonicates (3) and outer membrane proteins (11) of H. hepaticus have been used as ELISA antigens. In parallel with lesion development and serum alanine transaminase levels, antibodies have been first detected in naturally infected animals (of susceptible strains from enzootically infected colonies) at about 6 months of age. These ELISA titers increase in O.D. intensity as the mice age, reaching maximal levels at about 12-18 months of age and more so in males than females (3). The ELISA using H. hepaticus antigens appeared quite specific, recognizing antibodies to H. hepaticus, but not to H. bilis or H. muridarum (11).
The original diagnosis of murine helicobacteriosis was based on histopathologic criteria of silver staining spiral bacteria in clinically incidental cases of hepatitis (2). Histopathology remains an important tool in differentiating causes of hepatitis in laboratory mice, and the finding of silver staining spiroform bacteria, particularly in bile canaliculi, is considered diagnostic. The method suffers for adoption as a useful screening device because of its insensitivity (large numbers of bacteria required for microscopic demonstration) and because, even in susceptible strains, liver lesion development is quite variable [and may not exceed 10% of infected individuals (1)] and occurs late in the course. Resistant strains do not develop lesions to focus the microscopic search for organisms. Additionally, the method does not allow discrimination between Helicobacter species and, in intestinal sections, cannot discriminate between Helicobacter sp. and spiroform commensals. On balance, microscopic histopathology is useful in describing morphologic changes attributable to Helicobacter sp. and for confirming clinical diagnosis in lesioned animals, but not as a screening test.
Microbiologic isolation of Helicobacter hepaticus was initially conducted by inoculation of moist Columbia blood agar plates with material from lesioned livers, followed by anaerobic incubation. Subsequent refinements of the method involved use of either moist blood agar or Brucella agar plates with TVP (trimethoprin, vancomycin, polymixin) to inhibit contaminating bacteria from clinical materials (tissue or fecal emulsions) and a specialized microaerophilic gas incubation environment (N2, H2, CO2 in a ratio of 90:5:5) (12). It was determined that unwanted overgrowing microbial contamination could be further reduced by passing the clinical inoculum through a 0.45um filter that would allow passage of H. hepaticus, but retain many contaminants. Later, it was found that the same principle using a 0.65um filter would allow passage of H. hepaticus as well as the larger helicobacters (H. bilis, H. trogontum and H. rappini) ,which are retained by a 0.45Ý filter, (3). Isolated Helicobacter cultures are identified by their size (filter passage), colonial morphology of spreading growth films with spiroform Gram negative bacteria that are urease, catalase and oxidase positive. Some species, e.g. H. rodentium are urease negative, but urease production has not been developed as a useful feature for clinical diagnosis of murine infections (as it has for human H. pylori infections). Isolation in culture is required to enable speciation of isolates on the basis of biochemical criteria [(see Table in ref. (3)]. Culture has been widely used as a screening method, but important limitations include the obscuring presence of contaminating microbial overgrowth and low bacterial numbers, both of which bear on the reliability of negative results. An additional limitation is the 2-3 week incubation/holding period before cultures can be discarded as negative.
As indicated earlier, serology has received some attention as a screening method, but like cultural isolation, has a number of important limitations that inhibit its endorsement as a generally useful diagnostic method. Since antibody levels to Helicobacter are generally proportional to the microbial challenge, positive serology is probably reliable as an indicator of exposure to the antigen used in the test. The problem is that negative serology is not a reliable result for any of a number of reasons, including: a) innate host resistance that prevents lesion development or significant antigenic challenge, b) initially low numbers of organisms in the enteric reservoir, c) serum samples drawn early in the course before development of detectable titers and, perhaps most importantly, d) infection by Helicobacter species that induce antibodies not recognizing antigens used in the test, i.e., issues of specificity.
The polymerase chain reaction (PCR) has been developed (13) as the most useful single screening test for detection of murine helicobacters in clinical materials including tissue and fecal specimens, and retrospective material including wet fixed tissues, paraffin imbedded tissues, and even stained histologic preparations. The method is based on the detection of unique and subsequently amplified 16S rRNA gene sequences of the organism extracted from the sample material. The procedure is specific to the one or more Helicobacter species with sequences specified by the primers used for amplification and is not complicated by the presence of other microorganisms in the sample material. The test is rapid (same day) and by use of restriction endonucleases can be used to speciate Helicobacter DNA detected in generic tests (14). Perhaps the most important property of the method is extreme sensitivity for detection of low numbers of Helicobacter organisms in sample specimens. In direct comparisons, PCR detected as positive at least 30-50% more specimens found negative from animals also tested by culture and/or histopathology or electron microscopy (12, 15). Furthermore, the method permits pooling of samples without loss of sensitivity thus economizing on the cost of testing. Under carefully controlled circumstances, PCR is the method of choice for determination of the Helicobacter carrier status of given rodent colonies.
Because of institutional concerns about the potential of Helicobacter infections to spread within both breeding and user facilities and to clinically impact research programs, treatment modalities have been explored as a means of eradicating Helicobacter from infected carriers, or for reducing the clinical manifestation of helicobacteriosis in mouse colonies. It has been shown that two week treatments of amoxicillin alone, dosed via the drinking water, were effective in eliminating (or preventing) H. hepaticus infection in weanlings, but not in older mice with established enteric colonization (16). Triple treatment with combinations of amoxicillin, metronidazole and bismuth (known to eradicate H. pylori in humans and H. mustelae in ferrets) given by gavage 3 times daily for 2 weeks appeared effective in eradicating H. hepaticus infections in 6-8 month old susceptible inbred mice (17), but the labor intensive nature of the dosing regimen limits usefulness of this individual treatment. More promising was the ad lib oral treatment with amoxicillin triple treatment formulated into (now commercially available) flavored dietary wafers used to eradicate H. hepaticus in 6-10 month old naturally infected, susceptible inbred mice (18). As with the gavage method, the dietary wafer appears most useful to clear small defined pilot groups of the infection. The general problem of antibiotic treatments to suppress rather than fully eradicate, however, potentially sets the stage for rebound of residual Helicobacter populations after withdrawal of treatment. An additional area of concern is the uncertain susceptibility of other Helicobacter species to the treatment modes discussed above with H. hepaticus. It has been reported (6) that although diarrheas due H. bilis/H. rodentium infections of SCID mice could be clinically ameliorated, the Helicobacter infections were not eradicated after 2 week treatments with dietary wafers.
Not a great deal is known about other means to eradicate Helicobacter infection. Although essentially anecdotal and unreported, the successful eradication of H. hepaticus infection at the FCRC production and user colonies was oriented to principles familiar with eradication of other highly infectious murine pathogens: depopulation of selected units, thorough sanitization of these empty rooms and repopulation with same strain stocks rederived by either cesarean section or embryo transfer or H. hepaticus-free stock brought in from outside sources. Stringent maintenance regimens for admission of personnel and reprocessed equipment and supplies were put in place to prevent reinfection of the clean stock.
In attempting to weigh the pathogenic significance of rodent helicobacteriosis, there are a number of qualifiers that bear on the subject. As a general statement, it would seem warranted to regard the rodent helicobacters as essentially opportunistic enteric bacteria with only low grade potential to cause disease, i.e., clinical signs and lesions in outbred immunocompetent rodent stocks. That said, it is also the case that there is an accumulating series of reports detailing enterohepatic disease syndromes in variably immunocompetent, but susceptible inbred mice, as well as in immunodeficient and transgenic mouse strains. Complicating the issue is the proliferating diversity of microbial species and strains within the genus Helicobacter. As summarized in Table 1, it can be seen that so far at least 5 species have been characterized as indigenous to mouse hosts, 2 species from the hamster and 1 rat Helicobacter. To date, Helicobacter isolates have not been reported from the guinea pig, gerbil or rabbit. In general, the Helicobacter species are fairly host specific, but not entirely. For example, both H. bilis and H. muridarum (of the mouse) have been isolated from rats (3), and H. bilis has been reported as a cause of enteric disease in athymic rats (7). Most laboratories working with these agents find occasional isolates that don't neatly fit one of the characterized species, thus we should expect that not all of the variants have been carefully speciated as yet.
The Helicobacter species also vary in pathogenicity, even in susceptible hosts. Thus, H. bilis and H. hepaticus have been documented as pathogens on a number of occasions in a wide range of mouse strains and should therefore be regarded as frank pathogens of susceptible hosts. On at least two occasions (19, 20) H. muridarum has been microscopically associated with a mild gastriris in naturally infected mice.
There is no strong convincing evidence, at present, to indict H. rappini , H. rodentium and H. trogontum as rodent pathogens under circumstances of natural infection. The same assessment applies to H. cinaedi (in hamsters), but the status of H. cholecystis is uncertain at present. H. cholecystis was initially isolated from the gall bladders of hamsters with cholangitis and pancreatitis (21), but establishment of its pathogenicity via Koch's postulates have not been reported. Nonetheless, the (tenuous) designation of "nonpathogens" should be cautiously applied. Given the propensity of this genus to cause enterohepatic disease in such a widely diverse range of rodent and nonrodent mammalian hosts, it may well only be that the right mix of agent/host/milieu has not been encountered to eventuate (and be recognized) as a cause of clinical disease. A good example of this potential was reported recently in an article describing diarrhea with hypertrophic colitis in scid mice with dual H. bilis and H. rodentium infection, in which it was stated that subsequent experimental infections of H. rodentium alone did cause typhlocolitis in A/J mice (6).
There has been a curious reluctance on the part of some facility managers to incorporate the mounting tide of information about the rodent helicobacters into procurement standards, based in part, perhaps, on uncertainty as to the realistic pathogenic potential of such infections. We are probably past the point now where decision making about the consequences of helicobacteriosis can be reasonably deferred. To that end, the following might be considered as guidelines, at this point in time, for institutional procurement and maintenance programs:
Both H. hepaticus and H. bilis should be treated as pathogens unacceptable for either incoming or resident mouse populations. Even if the contamination is thought limited to resistant stocks or certain rooms, there is now such ample evidence of cage-to-cage and room-to-room transmission based on sentinel data (27), that the hazard of introduction of these agents to susceptible rodent strains, if present in the facility, needs to be realistically dealt with.
H. rappini, H. muridarum and H. rodentium should probably be regarded as undesirable contaminants of either incoming mice or resident populations. Their presence might be considered conditionally acceptable for some programs. However, programs with substantial populations of susceptible immunodeficient, inbred and/or transgenic mouse strains should probably list all members of the genus Helicobacter on the agent profile used as policy to define agents to be excluded from the facility.
Although contamination of hamsters with H. cinaedi may have zoonotic potential (3), the status of H. cinaedi and H. cholecystis in hamsters and H. trogontum in rats should probably be dealt with as above for the nonpathogenic mouse helicobacters. In the absence of heritable immunodeficiency or protocols which render the animals immunodeficient, these agents should be regarded as conditionally acceptable, but undesirable contaminants.
|* Y = Yes, associated with reported enterohepatic disease syndromes in host species
M= Maybe, several reports of microscopic gastritis (H. muridarum), cholangitis (H.cholecystus) and one report of typhlocolitis in A/J mice ( H. rodentium).
N = No, so far not reported as a natural cause of disease in host species.
** At present still formally designated Flexispira rappini