Suboptimal ability of dirty-bedding sentinels to detect Spironucleus muris in a colony of mice with genetic manipulations of the adaptive immune system.

Spironucleus muris (Figures 1 and ​and2)2) is classified as a parasitic diplomonad flagellate in the family Hexamitidae. Originally described in 1881 and called Dicercomonas muris,19 the organism was renamed, first as Hexamita muris44 and later as Spironucleus muris.8 In 1969, an article29 was published implicating Spironucleus as a cause of disease in mice; prior to that time, the protozoan was considered to be nonpathogenic. Other reports12,17,27,36,43 corroborating the parasite's ability to cause clinical illness quickly followed. In the 1970s and early 1980s, a considerable amount of research was done to characterize the parasite, and its presence was shown to interfere with immunology research in mice.6,7,21,34 Consequently, S. muris has been considered an unacceptable pathogen for research animals, and currently vendors and most rodent research facilities, including the National Institute of Allergy and Infectious Diseases, exclude this protozoan from their rodent colonies. Figure 1. Artist depiction of S. muris. The trophozoite is teardrop-shaped and has 6 anterior and 2 posterior flagella. The posterior flagella arise internally from the anterior region of the cell and emerge from the 2 subterminal openings or cytostomes on the ... Figure 2. Cross-section of a pyloric crypt containing numerous S. muris organisms with prominent paired anterior nuclei that give the protozoan a ‘2-eyed’ appearance. Hematoxylin and eosin stain; magnification, ×1000. Rodent colonies at the National Institute of Allergy and Infectious Diseases were monitored for protozoa by screening sentinel mice exposed to dirty bedding from research mouse cages. Sentinel mice were submitted to the National Institutes of Health, Division of Veterinary Resources, Animal Health Surveillance Laboratory approximately every 6 wk. Screening for intestinal protozoa was performed by direct examination of intestinal contents. In March 2006, 1 of 28 sentinel animals from 1 room of a facility was reported as positive for S. muris. This room had been depopulated and the research colonies embryo-rederived 3 y earlier to eradicate the protozoa. Therefore, this result generated great concern. Research use of the mice in the room involved extensive breeding, with a goal of having multiple targeted and nontargeted mutations of the immune system on single mice. Many of the strains in the room were not commercially available and required homozygous rederivation to preserve their engineered genetics. According to program standard operating procedure, the room was quarantined. S. muris infection was confirmed by testing a cagemate of the index positive sentinel animal. Subsequently cull mice from the research colonies were sent to the Health Surveillance Laboratory to determine the extent of infection; 93 of 267 mice (35%) submitted were found to be positive, with positive animals identified on all racks in the room. The researchers, dreading the lost research time involved with eliminating the parasite again, expressed concern about the inconsistency of the direct smear findings as well as the need to eradicate the organism. The direct smears showed positive and negative animals in the same cage. However, the investigators stated that sometimes the animals reported as positive were immunocompetent, whereas their immunocompromised cagemates reportedly were uninfected. In addition, the investigators’ expected research results, involving both T- and B-cell derangements,25,26 had not changed since the last room depopulation, nor had colony breeding parameters or incidence of illness in the various lines changed. Therefore the researchers questioned the reliability of using direct intestinal examination to identify the organism, as well as the conclusions reached in the early published articles about the organism's effect on animal health and immunology research. Cull mice from the involved room were studied to explore the apparent inconsistencies seen with direct intestinal examination for the parasite. Histopathology of the upper small intestine and gastric pylorus, a method considered to be superior to other diagnostic methods,32 was chosen as a secondary assessment method. An extensive literature review of S. muris was completed, and sentinel mice with variable durations of exposure to dirty-bedding were tested for the protozoa in an effort to improve the ability of the program to detect S. muris. The rationale for the redesigned program plan is presented.