There is little information concerning Helicobacter
status in noncommercial animal facilities. Data from Japan showed that 27 out of 47 (57.5%) sentinel mice obtained during 1998 and 1999 from universities, breeding companies, pharmaceutical companies, and national research institutions were contaminated with Helicobacter
). A Korean study revealed Helicobacter
infection in only one out of 24 mouse strains that were housed in SPF facilities (29
). A European study reported a prevalence of Helicobacter
infection of 88.9% (eight of nine strains) in mice (25
). Here, we found a comparable high prevalence rate of 87.5% in mice reared in different caging systems in an SPF facility.
Interestingly, none of the mice in this study was infected by H. bilis
and only six animals harbored H. hepaticus
. In earlier studies H. bilis
and H. hepaticus
were identified as the most common Helicobacter
species in laboratory mice (27
). This observation led to the addition of H. bilis-
and H. hepaticus
-specific PCR assays to the recommended screening protocol for SPF rodents (25
). The relatively low prevalence of both bacteria is presumably due to the use of these screening procedures.
The most frequent Helicobacter
species that we found in this study were H. ganmani
and the Helicobacter
species represented by isolate MIT 98-5357. H. ganmani
and the Helicobacter
species represented by isolate MIT 98-5357, as well as the Helicobacter
species represented by isolate hamster B, H. typhlonicus
, and the other Helicobacter
species we detected, are widely uncharacterized and thus no species-specific PCR assays are available for health screening of mice. Therefore, these rather uncommon Helicobacter
species may be missed by the routine procedures used for health screening of SPF mice and therefore explain the relatively high prevalence of these rare species in Helicobacter
-infected mice. Future studies will show whether this is a general trend or specific for the SPF facility tested. Moreover, our study clearly shows that the Helicobacter
species represented by isolate hamster B, which was recently detected in Syrian hamsters (35
), is also able to infect mice.
In order to avoid the spread of Helicobacter
infections in an animal facility, it was important to elucidate the transmission route. Recently, it was shown that H. hepaticus
can be transmitted by contaminated bedding (19
). Therefore, accidental bedding contamination could be a possible reason for Helicobacter
transmission. However, this possibility was considered unlikely based on the high hygiene standard in our facility. Thus, it was necessary to examine the possibility of alternative transmission routes.
One important observation is that we identified mice from commercial breeding facilities as potential infection sources. One mouse strain of a commercial vendor carried a Helicobacter infection, although the accompanying health report attested that the animals were Helicobacter negative as tested by PCR (Table ). Since animals from commercial SPF facilities are usually not taken into quarantine rooms, unrecognized infections in these animals can introduce Helicobacter infections into the animal facility. It is therefore advisable to keep animals from commercial vendors in quarantine rooms until the health status of every shipment is confirmed.
As study 1 showed, Helicobacter
infections can be effectively prevented by harboring mice in individually ventilated cages even if infected mice were reared in neighboring cages and if the same laminar flow hood was used to change the cages of uninfected and infected animals. However, since individually ventilated cages are expensive, immunocompetent wild-type mice are frequently harbored in open-air cages. Our studies 3 and 4 showed that mice reared in open-air cages were very likely to get infected, but for a certain time period not all mice are infected. This fact is important because Helicobacter
infections in wild-type mice often lack clinical signs (39
). Therefore, it is not possible to select diseased sentinel mice for Helicobacter
screening. By selecting sentinel mice at random, the chance of detecting EHS-infected animals is decreased and as a consequence Helicobacter
-infected colonies may remain unrecognized. If such wild-type animals with unrecognized Helicobacter
infections are then used as sentinels or for backcross purposes, these animals are transferred into individually ventilated cages together with Helicobacter-
free knockout animals. As study 2 indicated, cage mates then get infected within 2 weeks in 100% of the cases.
We have often observed that the mice in open-air cages distributed stool pellets and contaminated bedding around their cages onto the floor and into neighboring cages. This can be a significant amount of material, which is easy to observe by inspecting the floor next to racks with open-air cages after a weekend if the floor is not cleaned every day. Mice are coprophages. The high rate of Helicobacter transmission between neighboring open-air cages suggests that stool pellets which were transferred in this way between cages are ingested by neighboring mice, which in turn get infected. As study 5 showed, the use of filter tops on conventional cages to prevent the spread of stool and contaminated bedding to neighboring cages was sufficient to block the transmission of EHS. These findings strongly support a fecal-oral spread of EHS in mice.
Taken together, infections with EHS can be introduced into an SPF facility and in turn may spread among animals. In order to maintain SPF animals free of Helicobacter infections, we recommend the following measures: use of a group-specific PCR assay to test animals regularly for Helicobacteraceae; keeping mice which are introduced into the facility in individually ventilated cages until the Helicobacter status of every shipment is confirmed and, whenever feasible, keeping mice in individually ventilated cages or cages with polycarbonate filter tops; and checking mice which are placed as sentinels or mice used for backcrossing experiments to ensure that they are Helicobacter negative.