Germ-free mice are used to examine questions about the role of the gut microbiota in development of diseases. Generally these animals are maintained in semi-rigid or flexible-film isolators to ensure their continued sterility or, if colonized with specific microbiota, to ensure that no new species are introduced. Here, we describe the use of a caging system in which individual cages are hermetically sealed and have their own filtered positive airflow. This isopositive caging system requires less space and reduces animal housing costs. By using strict sterile techniques, we kept mice germ-free in this caging system for 12 weeks. We also used this caging system and approach to conduct studies evaluating a) the stability of the microbiome in germ-free mice receiving a fecal transplant and b) the stability of dietary-induced microbiota changes in fecal-transplanted mice. As has been shown in fecal transfer studies in isolators, we found that the transferred microbiota stabilizes as early as 2 weeks post transfer although recipient microbiota did not completely recapitulate those of the donors. Interestingly, we also noted some sex effects in these studies indicating that the sex of recipients or donors may play a role in colonization of microbiota. However, a larger study will be needed to determine what role, if any, sex plays in colonization of microbiota. Based on our studies, an isopositive caging system may be utilized to test multiple donor samples for their effects on phenotypes of mice in both normal and disease states even with limited available space for housing.

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A 225-kDa Babesia bovis protein occurs on the cytoplasmic side of infected-erythrocyte membranes. Here it is demonstrated that the 225-kDa protein localizes to spherical-body organelles of merozoites. Organelles consistent in size and shape with spherical bodies were isolated between 1.17 and 1.21 g/cm 3 in a sucrose density gradient. Organelles consistent with rhoptries and micronemes were also present in fractions from 1.17 to 1.19 g/cm 3. Antisera generated by immunizing mice with the fraction (1.20 to 1.21 g/cm 3) most enriched for spherical bodies reacted predominantly with spherical bodies in B. bovis merozoites. A monoclonal antibody generated from this immunization (70/97.14) recognized an epitope that occurs in the repeat region of the 225-kDa protein (now referred to as SBP2). Monoclonal antibody 70/97.14 bound to merozoite spherical bodies, vesicles in infected-host cytoplasm, and the cytoplasmic face of the infected-erythrocyte membrane. These results indicate that spherical-body proteins become associated with the host membrane via transport through the erythrocyte cytoplasm after intracellular invasion. Babesia bovis is a tick-borne apicomplexan parasite that invades, multiplies within, and eventually causes lysis of bovine erythrocytes. Identification of parasite proteins that mediate invasion and erythrocyte destruction may facilitate development of strategies to control this parasite. Apicomplexan parasites produce several proteins that interact with host cell membranes. These proteins are contained within the apical complex which includes the rhoptries, micronemes, dense granules, and spherical bodies. Examples include dense-granule proteins of Toxoplasma gondii that become associated with the parasitophorous vacuole membrane and/or microtubular network shortly after invasion (8) and a 155-kDa Plasmodium falciparum dense-granule protein (RESA) (4) that interacts with spectrin on the cytoplasmic face of ringinfected erythrocytes (12). Rhoptry, dense-granule, and microneme proteins also interact with host cell membranes at the time of invasion by P. falciparum (43) and P. knowlesi (50) or with erythrocyte knobs in the case of P. brasilianum (51). Thus, the functions of apical-complex organelle components include host cell interaction and modification for parasite invasion and/or intracellular growth. Previous studies (28, 41) identified a 225-kDa B. bovis protein that localizes to a focal region of the merozoite and the cytoplasmic side of the infected-erythrocyte membrane. The 225-kDa protein gene belongs to a larger gene family, and the protein was contained in a B. bovis fraction that induced protective immunity against the parasite (10, 24). The 225-kDa protein is conserved among geographically diverse isolates of B. bovis. Conservation includes an epitope encoded by a 73amino-acid repeat and recognized by monoclonal antibody (MAb) 23/8.34 (28). Therefore, it is of interest to determine the intraparasitic and intraerythrocytic locations in the parasite and host cell, respectively, possible fun...

Reliable detection of unwanted microbial agents is essential for meaningful health monitoring in laboratory animal facilities. Most rodents at our institution are housed in IVC rack systems to minimize aerogenic transmission of infectious agents between cages. The most commonly used rodent health monitoring systems expose live sentinel rodents to soiled bedding collected from other rodent cages on IVC racks and subsequently test these soiled-bedding sentinels for evidence of infection with excluded agents. However, infectious agents might go undetected when using this health surveillance method, due to inefficient organism shedding or transmission failure. In 2016, our institution switched the health monitoring methodology for the majority of our SPF rodent colonies to real-time PCR testing of environmental samples collected from the exhaust plenums of IVC racks. Here we describe our rationale for this conversion, describe some interesting health monitoring cases that arose soon after the conversion, and discuss a potential problem with the conversion—residual nucleic acids. We compared cost and implementation effort associated with 2 sampling methods, sticky swabs and in-line collection media. We also compared the ability of these 2 sampling methods to detect 2 prevalent microbes in our facilities, Helicobacter and murine norovirus. Our institution-wide switch to health monitoring by real-time PCR assay of exhaust air dust samples thus far has provided a sensitive, simple, and reliable approach for maintenance of SPF conditions in laboratory rodents and has dramatically reduced the use of live sentinel animals.

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