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Parasitol., 89(2), 2003, pp. 391–393  American Society of Parasitologists 2003 Cryptosporidium parvum Infection in Gene-Targeted B Cell–Deficient Mice Wangxue Chen, James A. Harp*, and Allen G. Harmsen†, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, Canada K1A 0R6; *Periparturient Diseases of Cattle Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010-0070; †Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717. e-mail: wangxue.chen@nrc.ca ABSTRACT: The importance of B cells in host resistance to, and recov- ery from, Cryptosporidium parvum infection was examined in gene- targeted B cell–deficient (MT-/-) mice. Neonatal MT-/- mice in- fected with C. parvum at 5 days of age completely cleared the infection by day 20 PI. The kinetics of infection and clearance were similar to those seen with age-matched C57BL/6 control mice. Furthermore, B cells were not required to clear existing C. parvum infection in adult mice. Reconstitution of persistently infected Rag-1-/- adult mice with spleen cells from MT-/- donor mice resulted in significant reduction of infection, as in the results seen with spleen cells from C57BL6 do- nors. These findings indicate clearly that B cells are not essential for host resistance to, and recovery from, C. parvum infection in mice. Cryptosporidium parvum is an important cause of intestinal infections in immunocompromised humans, especially in acquired immunodefi- ciency syndrome (AIDS) patients, whereas the infection is usually self- limiting and probably often undiagnosed in immunocompetent hosts (Laughon et al., 1991; Zu et al., 1992). The mechanism of host resis- tance to, and recovery from, C. parvum infection remains poorly de- fined. Recent clinical and experimental studies show that both CD4 + cells and interferon (IFN)- are necessary for host resistance to C. par- vum infection, whereas other components, including nitric oxide and interleukin (IL)-4, also may play a role (Ungar et al., 1991; Chen et al., 1993; Perryman et al., 1994; Aguirre et al., 1998; Leitch and He, 1999; White et al., 2000). However, it is not known what other immune mech- anisms are involved in host defense against C. parvum infection. The fact that C. parvum infection is self-limiting in nearly half the AIDS patients, with CD4 + T cell counts in the circulation below 200 cells/ mm 3 (Flanigan et al., 1992), suggests that factors other than T cells also play a role in controlling C. parvum infection. Indeed, the functions of B cells and nonlymphoid cells in AIDS patients are also abnormal (Bowen et al., 1985), and this in turn could contribute to their increased susceptibility to C. parvum infection. The importance of B cells in host defenses against C. parvum has not been well established, and the role of immunoglobulins in protective immunity against C. parvum remains controversial (McDonald and Ban- croft, 1998). Many groups have examined the kinetics of the production of C. parvum–specific antibodies of different classes in the serum and mucosal secretions and their association with oocyst shedding in in- fected humans, mice, cattle, and sheep (reviewed in McDonald and Bancroft, 1998). Whereas some have found an association between de- clining oocyst shedding and rising titers of specific IgA and IgM, others have failed to establish such an association. Several studies, however, suggest that humoral immunity has little protective effect on C. parvum infection (Zu et al., 1992; McDonald and Bancroft, 1998; de Graaf et al., 1999; Weiner et al., 1999). The presence of high titers of C. parvum– specific antibodies in almost all the AIDS patients with persistent symp- tomatic cryptosporidiosis further implies that humoral immunity alone is not sufficient to resolve the infection (Soave and Johnson, 1988; Zu et al., 1992; Cozon et al., 1994). Direct evidence that B cells and an- tibody are not required for anti–C. parvum immunity was obtained in the study by Taghi-Kilani et al. (1990), who showed that neonatal mice depleted of B cells by anti- antibody treatment were able to control C. parvum infection as efficiently as control mice. Taken together, these findings favor the notion that humoral immunity is not a requirement for clearing infection, and by itself is not sufficient to control enteric infection with C. parvum. It is worth noting that the mice that were rendered B cell deficient by anti- antibody treatment (Taghi-Kilani et al., 1990) contained small numbers (5%) of sIg-expressing B cells in the spleen and lymph nodes and developed low amounts of serum and gut Ig of all isotypes. In addition, the treatment with anti- antibodies might have affected cells other than B cells, making it difficult to assess these effects in an un- equivocal manner (Langhorne et al., 1998). Gene-targeted disruption of the IgH locus produces mice completely devoid of both antibodies and B cells (Kitamura et al., 1991). In the present study, we examined the requirement for B cells in resistance to initial C. parvum colonization by using these gene-targeted B cell–de- ficient mice (MT-/- mice). We also used these mice in splenocyte transfer experiments to determine the importance of B cells in the res- olution of an established C. parvum infection. The results reported here showed that B cells are not essential for either resistance to C. parvum colonization or resolution of an established C. parvum infection. C57BL/6J (B6) and C57BL/6J Igh-6 tm1 Cgn (B cell deficient, MT-/ -) mice were obtained from the Trudeau Institute Animal Breeding Facility (Saranac Lake, New York). C57BL/6J Rag-1 tm1 mom (Rag-1-/-) mice were purchased from Taconic Farms, Germantown, New York. Mice were housed in microisolator cages containing sterilized food and water and were shown to be free of an extensive series of common murine pathogens including C. parvum. Age- and sex-matched mice were used in all the experiments. Purified C. parvum oocysts, free of bacterial contamination, were prepared from feces collected from calves experimentally inoculated with C. parvum (Iowa isolate) by a method described previously (Harp et al., 1992). Mice were inoculated orally with a single dose of oocysts, as indicated in the text, in 0.2 ml of 0.15 M phosphate-buffered saline (pH 7.2) by using a 19g gavage needle. To determine infection after C. parvum challenge, fecal pellets were collected from individual mice and smeared onto glass slides. These smears were stained with carbol-fuchsin and examined for the presence of C. parvum oocysts as described previously (Waters and Harp, 1996). Mice were killed by CO 2 asphyxiation at the predetermined times. The ileum, cecum, and proximal colon were removed from each mouse and fixed by immersion in 10% neutral buffered formalin. After fixation, the tissue was embedded in paraffin. Sections of 4-m thickness were cut, stained with hematoxylin–eosin (HE), and examined microscopi- cally for C. parvum. An infectivity score of 0–3 was assigned to each