© 2005 Blackwell Publishing Ltd 17 Parasite Immunology , 2005, 27, 17–28 Blackwell Publishing, Ltd. ORIGINAL ARTICLE Responses to Cryptosporidium in IL-12p40 KO mice Mucosal cytokine and antigen-specific responses to Cryptosporidium parvum in IL-12p40 KO mice H. N. EHIGIATOR, 2 P. ROMAGNOLI, 1,2 K. BORGELT, 1 M. FERNANDEZ, 1 N. MCNAIR, 1 W. E. SECOR 3 & J. R. MEAD 1,2 1 Veterans Affairs Medical Center, Decatur, Georgia, USA, 2 Emory University School of Medicine, Department of Pediatrics, Atlanta, Georgia, USA and 3 NCID, Centers for Disease Control & Prevention, Atlanta, Georgia, USA SUMMARY Studies of cellular immune responses to Cryptosporidium parvum have been limited in part by lack of suitable animal models. IL-12p40 –/– mice are susceptible to initial infection with C. parvum but recover within 2 weeks, rendering the ani- mals resistant to reinfection. Because the host responses that determine duration and severity of primary infection are not yet understood, we studied the cellular immune response to primary infection with C. parvum in IL-12p40 –/– mice and also explored possible mechanisms for this response. Female IL- 12p40 –/– mice were inoculated with 10 000 oocysts. Uninfected age-matched mice served as controls. At different time inter- vals following exposure to oocysts, mice were sacrificed and their intestine, spleen, and mesenteric lymph node tissues were harvested. Cellular immune responses to C. parvum were characterized. Infection of IL-12p40 –/– mice induced changes in the gene expression of the cytokines IFN-γ, IL-4, IL-15, IL-18, TNF-α and TGF-β during primary infection. There was also a significant increase in total numbers of lymphocytes and CD19/CD62L-expressing cells in mesenteric lymph nodes. These MLN cells exhibited increased antigen-specific prolif- eration and cytokine production (IL-6 and IFN-γ) levels when stimulated in vitro. These observations delineate the cellular immune responses during acute C. parvum infection of the IL-12p40 –/– mouse model. Keywords cellular response, Cryptosporidium parvum, cytokine, IL-12p40-knockout, mouse, real-time PCR INTRODUCTION Cryptosporidium parvum, an opportunistic parasitic proto- zoan with worldwide distribution, is a major cause of diar- rhoeal illness in children and individuals with compromised immune systems, such as in people with acquired immuno- deficiency syndrome (1,2). Several outbreaks with morbidity and mortality have been associated with C. parvum infec- tion, the largest of which occurred in Milwaukee in 1993 (3,4). Infections with C. parvum are generally self-limiting in healthy individuals, but immunocompromised individuals experience more severe and extended symptoms that can be life-threatening (2,3,5,6). The host defence mechanisms responsible for limiting Cryptosporidium infections are not fully understood. How- ever, evidence from several murine, bovine, and human studies suggest an important role for cell-mediated immune responses, including T cell-derived cytokines, in the recovery from Cryptosporidium infection (7–9). Several studies have reported the induction of a number of cytokines such as IL- 1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-15, IFN-γ, TNF-α, TGF- β, and the chemokine RANTES following murine, bovine, human, and cell line infections with Cryptosporidium (10– 19). Of these cytokines, IFN-γ has been shown to be essen- tial in the host protective immune response to C. parvum infection. This observation is clearly demonstrated in stud- ies showing that infection of IFN-γ knockout (IFN-γ-KO) mice on a C57BL/6 background resulted in severe infections with high levels of oocyst shedding, resulting in death (13,18,20 –22). Studies of the protective cellular immune responses to C. parvum have been limited by the lack of a suitable adult immunocompetent reinfection model. A recent study by Campbell et al. (23) demonstrated C. parvum infection in mice with targeted mutation in the IL-12p40 gene. These mice develop a robust infection from which they recover within 14–16 days. This model, unlike the IFN-γ-KO model, allows for the study of protective immunity as the mice still Correspondence: Dr Jan R. Mead, Veterans Affairs Medical Center, Medical Research 151, 1670 Clairmont Road, Decatur, GA 30033, USA (e-mail: jmead@emory.edu). Received: 27 August 2004 Accepted for publication: 2 February 2005