Profiles of healing and nonhealing Cryptosporidium parvum infection in C57BL/6 mice with functional B and T lymphocytes: the extent of gamma interferon modulation determines the outcome of infection.

This study describes healing and nonhealing models of Cryptosporidium parvum infection with adult mice that have functional T and B lymphocytes. In our nonhealing model, mice on a C57BL/6 background which have a targeted disruption in the gamma interferon (IFN-gamma) gene (GKO mice) are utilized. C. parvum-infected GKO mice shed extremely high levels of oocysts and displayed overwhelming infection of the entire small intestine. The majority of these mice succumbed within 2 to 3 weeks due to severe acute infection and profound mucosal destruction. In our healing murine model, C57BL/6J mice treated with a single injection of the neutralizing anti-IFN-gamma monoclonal antibody XMG 1.2 prior to infection were used. These mice developed two peaks of oocyst shedding but were ultimately free of parasites on day 30 of infection. Again, the small intestine was the primary site of infection. Mesenteric lymph node (MLN) cells isolated from C. parvum-infected nonhealing GKO mice proliferated and secreted interleukin 2 (IL-2) but not IFN-gamma or IL-4 in response to ex vivo restimulation with intact C. parvum sporozoites or a C. parvum sporozoite antigen preparation. In contrast, parasite-specific MLN cells isolated from healing C57BL/6J mice secreted IL-2 and IFN-gamma but not IL-4. These results suggest that IFN-gamma, either directly or indirectly, is important for resistance to and resolution of cryptosporidiosis. Moreover, these models now allow the analysis of parasite-specific cell-mediated and humoral mucosal immune responses to determine what constitutes protective immunity to C. parvum.     

The association between lung innate immunity and differential airway antigen- specific immune responses

The mechanisms involved in the differential regulation of airwayimmune responses in atopic versus non-atopic individuals arepoorly understood. In this study, the association between nonspecific immunity and the differential airway antigen-specificImmune responses was examined in a murine model. The disparityIn antigen-specific IgE and IgG2a productions between the twostrains of mice was observed to be significant. C57BL/6J micewere much more efficient than BALB/cJ mice in making IgE antibodyto Inhaled ovalbumin (OVA) antigen. On the contrary, BALB/cJmice did make more IgG2a antibodies than C57BL/6J mice to InhaledOVA. These findings suggest that in C57BL/6J mouse strain apredominant T_h 2 type of Immune response develops in responseto inhaled OVA antigen. In contrast, BALB/c mice mount a T_h1 type of immune response to aerosollzed OVA antigen. Furthermore,after lipopolysaccharlde (LPS) stimulation, the IL-12 mRNA expressionof lung-derived cells from BALB/cJ mice was higher than thatfrom C57BL/6J cells. However, the lung-derived cells of C57BL/6Jmice stimulated by LPS produced higher levels of IL-b and prostaglandinE than BALB/cJ lung-derived cells did. Therefore, our studydemonstrated that the difference of lung-derived cells in theirability to produce cytokine and prostaglandln between BALBIcJand C57BL/6J mice correlates well with the type of the airwayantigen-specific immune effector functions.     

Mouse strain differences in operant self-administration of ethanol

By the use of operant conditioning procedures, we determined whether mice from two strains, C57BL/6J and BALB/cJ, differ in ethanol-reinforced behavior. To establish ethanol as a reinforcer, drinking was induced by feeding mice prior to the 30-min experimental session. Initially mice received water and then a series of increasing ethanol concentrations (1, 2, 4, and 8%, w/v) in response to a lever press. The volume of liquid consumed per unit of body weight remained relatively constant across concentrations, and thus the amount of ethanol consumed (g/kg) increased with increases in ethanol concentration. At all concentrations the C57BL/6J mice drank larger volumes than the BALB/cJ mice. After shifting the time of feeding from before to after the session, the consumption of 8% ethanol declined 56% for the C57B/6J mice and 81% for the BALB/cJ mice. To determine if ethanol was serving as a reinforcer, behavior and intake were then compared during ethanol and water availability. The ethanol consumption of the C57BL/6J mice markedly exceeded the water consumption and resulted in blood ethanol levels of 199 ±27 mg/dl. These levels significantly exceeded the 8% ethanol intake of the BALB/cJ mice, while 0% intake was low and not significantly different between strains. The 8% ethanol drinking of the BALB/cJ mice did not differ from their water drinking. Thus, ethanol was established as a positive reinforcer for C57BL/6J mice but not for BALB/cJ mice.This research was supported in part by New Investigator Research Awards AA-06104 and AA-06924 to Frank R. George from the National Institute on Alcohol Abuse and Alcoholism and by Research Scientist Development Award DA-00007 to Richard A. Meisch from the National Institute on Drug Abuse.     

Genetic control of susceptibility to Porphyromonas gingivalis-induced alveolar bone loss in mice

Periodontal disease affects a large percentage of the human population. Resorption of the alveolar bone of the jaw is a pivotal sequela of periodontal disease, because this bone is the attachment site for the periodontal ligaments that anchor the teeth. Using a murine model in which alveolar bone loss is induced by oral infection with Porphyromonas gingivalis, a gram-negative bacterium associated with human adult periodontal disease, we provide evidence suggesting that susceptibility to such bone loss is a genetically determined trait. AKR/J, DBA/2J, and BALB/cByJ or BALB/cJ mice were highly susceptible, while A/J, A/HeJ, 129/J, SJL/J, and C57BL/6J mice were much more resistant. When susceptible BALB/cJ and BALB/cByJ mice were crossed to resistant strains, two patterns were observed. (BALBc/ByJ x C57BL/6J)F(1) offspring were susceptible, suggesting C57BL/6J has recessive resistance alleles, while (BALB/cJ x A/J)F(1) mice were all resistant, suggesting that A/J mice have dominant resistance alleles. These results suggest a tractable genetic basis for P. gingivalis-induced alveolar bone loss and open the possibility of exploiting the mouse model to identify loci important for host susceptibility and resistance to periodontal disease.     

The resistance of BALB/cJ mice to Yersinia pestis maps to the major histocompatibility complex of chromosome 17

Yersinia pestis, the causative agent of plague, has been well studied at the molecular and genetic levels, but little is known about the role that host genes play in combating this highly lethal pathogen. We challenged several inbred strains of mice with Y. pestis and found that BALB/cJ mice are highly resistant compared to susceptible strains such as C57BL/6J. This resistance was observed only in BALB/cJ mice and not in other BALB/c substrains. Compared to C57BL/6J mice, the BALB/cJ strain exhibited reduced bacterial burden in the spleen and liver early after infection as well as lower levels of serum interleukin-6. These differences were evident 24 h postinfection and became more pronounced with time. Although a significant influx of neutrophils in the spleen and liver was exhibited in both strains, occlusive fibrinous thrombi resulting in necrosis of the surrounding tissue was observed only in C57BL/6J mice. In an effort to identify the gene(s) responsible for resistance, we measured total splenic bacteria in 95 F(2) mice 48 h postinfection and performed quantitative trait locus mapping using 58 microsatellite markers spaced throughout the genome. This analysis revealed a single nonrecessive plague resistance locus, designated prl1 (plague resistance locus 1), which coincides with the major histocompatibility complex of chromosome 17. A second screen of 95 backcrossed mice verified that this locus confers resistance to Y. pestis early in infection. Finally, eighth generation backcrossed mice harboring prl1 were found to maintain resistance in the susceptible C57BL/6J background. These results identify a novel genetic locus in BALB/cJ mice that confers resistance to Y. pestis.     

Maternal influences on adult stress and anxiety-like behavior in C57BL/6J and BALB/cJ mice: a cross-fostering study

The quality of maternal care during early life has a dramatic impact on later stress reactivity and anxiety. Two inbred mouse strains, C57BL/6J and BALB/cJ, differ in levels of maternal care, stress reactivity, and anxiety-like behavior in adulthood. However, the relative contribution of early environmental factors and genetic predisposition to differences in these strains is not known. Maternal care, plasma corticosterone levels, emotionality, and hippocampal and paraventricular nucleus (PVN) glucocorticoid receptor mRNA levels were measured in adult C57BL/6J and BALB/cJ mice. Litters were then cross-fostered and anxiety-like behavior and stress reactivity was assessed in adulthood. Significantly less maternal care and elevated stress-induced corticosterone and emotionality was observed in BALB/cJ compared to C57BL/6J mice. Yet, no strain differences were found in hippocampal or paraventricular nucleus glucocorticoid receptor mRNA levels. Cross-fostering did alter anxiety-like behavior and basal corticosterone levels, which suggests that while genetic differences account for some of the variations between these two strains early rearing conditions also contribute.     

Differential immune response in two handled inbred strains of mice

C57BL/10J and BALB/cJ mice, outfostered at birth to C3H/2Ibg dams were subjected to handling on days 1 through 20 of life. Their plaque forming cell (PFC) response to sheep red blood cells as adults on day 5 post-immunization was compared to the PFC response in non-handled control mice. The PFC response of handled C57BL/10J mice was significantly suppressed compared to the PFC response in non-handled mice while the response of the handled and non-handled BALB/cJ mice was not significantly different.     

Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice.

Adult murine models of Cryptosporidium infection involving Cryptosporidium muris and C. parvum were used to study immunity to cryptosporidiosis in the mammalian host. Immunocompetent BALB/c or C57BL/6 mice developed a highly patent infection with the RN 66 strain of C. muris but overcame the infection and were immune to reinfection. In contrast, severe combined immunodeficiency (SCID) mice or nude mice had a chronic infection lasting at least 109 days. The development of the C. muris infection appeared to be confined to the gastric epithelium in immunocompetent and immunocompromised mice. SCID mice injected intraperitoneally with histocompatible spleen or mesenteric lymph node cells from uninfected BALB/c mice were able to recover from the C. muris infection. The protective effect of donor spleen cells was not reduced by depletion of the B cell population but was significantly reduced by depletion of Thy.1 cells. Treatment of C57BL/6 or BALB/c mice during infection with a gamma interferon-neutralizing monoclonal antibody, but not a tumor necrosis factor-neutralizing monoclonal antibody, resulted in a significant increase in oocyst production. In the C. parvum model, a severe and eventually fatal chronic infection with a cervine isolate was established in SCID mice, with parasitization occurring in the ileum, cecum, and colon. SCID mice injected with unprimed BALB/c spleen cells prior to inoculation of C. parvum oocysts were resistant to infection. These results suggested that the two animal models should be valuable in the study of immunity to cryptosporidial infection.     

Maternal influences on adult stress and anxiety‐like behavior in C57BL/6J and BALB/CJ mice: A cross‐fostering study

The quality of maternal care during early life has a dramatic impact on later stress reactivity and anxiety. Two inbred mouse strains, C57BL/6J and BALB/cJ, differ in levels of maternal care, stress reactivity, and anxiety‐like behavior in adulthood. However, the relative contribution of early environmental factors and genetic predisposition to differences in these strains is not known. Maternal care, plasma corticosterone levels, emotionality, and hippocampal and paraventricular nucleus (PVN) glucocorticoid receptor mRNA levels were measured in adult C57BL/6J and BALB/cJ mice. Litters were then cross‐fostered and anxiety‐like behavior and stress reactivity was assessed in adulthood. Significantly less maternal care and elevated stress‐induced corticosterone and emotionality was observed in BALB/cJ compared to C57BL/6J mice. Yet, no strain differences were found in hippocampal or paraventricular nucleus glucocorticoid receptor mRNA levels. Cross‐fostering did alter anxiety‐like behavior and basal corticosterone levels, which suggests that while genetic differences account for some of the variations between these two strains early rearing conditions also contribute. © 2005 Wiley Periodicals, Inc. Dev Psychobiol 48: 95–96, 2006.     

Cytotoxic effects of natural killer cells have no significant role in controlling infection with the intracellular protozoon Eimeria vermiformis.

The course of infection with Eimeria vermiformis in C57BL/6J; NK cell-defective C57BL/6J bg/bg; BALB/c; T-cell-defective BALB/c nu/nu; and T-cell-, B-cell-, and NK cell-defective BALB/c x C57BL/6 scid/scid bg/bg mice was monitored. For young C57BL/6J mice, the bg/bg mutants consistently produced fewer oocysts than the controls; there were no differences between older mice of these strains. Wild-type BALB/c mice were more resistant to infection than the nu/nu and scid/scid bg/bg mutants, but there was no difference between the mutants. Treatment of BALB/c mice with poly(I.C) had no effect on the course of infection. These findings confirm the ineffectiveness of NK cells in this system.     

Differential Effects of Infection with a Babesia-Like Piroplasm, WA1, in Inbred Mice

A newly identified intraerythrocytic Babesia-like organism, WA1, and its relatives were recently shown to be infectious for humans in the western United States. The purpose of the present study was to determine the susceptibilities of selected mouse genotypes to WA1 infection in an attempt to develop a murine model of the human disease. Several mouse strains were inoculated intraperitoneally with various passages of WA1-parasitized erythrocytes. Parasitemia was evaluated by blood smears and by PCR with blood samples collected at various intervals after inoculation. Hematologic parameters were monitored in blood samples at all intervals. C57BL/6 and C57BL/10 mice exhibited mortality rates of <10%. BALB/cJ, CBAJ, and 129/J mice had higher peak parasitemias than did C57BL mice, with mortality rates of 40, 50, and 50%, respectively. A/J, AKR/N, C3H, and DBA/1J mice also had higher peak parasitemia and mortality rates (>95%). An F₁ cross of C57BL/6 (resistant) and C3H.RKK (susceptible) mice had a mortality rate similar to that of the resistant parental strain. Histopathology of BALB/cJ and C3H mice at 9 and 14 days after inoculation revealed erythrophagocytosis and deposition of an iron-negative pigment in multiple organs. Morbidly ill C3H mice at 14 days had severe pulmonary edema, hemoglobinuria, and glomerulonephritis.     

Genotype-dependent characteristics of behavior in mice in cognitive tests. The effects of Noopept

Male C57BL/6J, BALB/c, and DBA/2J mice showed differences in their abilities to perform two cognitive tests. C57BL/6J mice had good learning ability and memory trace retention (at 10 days) in a simplified Morris maze, while BALB/c mice had low levels of memory trace retention and DBA/2J mice had low learning ability in this test. I.p. administration of the nootropic agent Noopept (GVS-111, N-phenylacetyl-L-prolylglycine ethyl ester) at a dose of 0.5 mg/kg 15 min before the start of the test induced significant improvements in long-term memory in this test in BALB/c mice but no further improvement in C57BL/6J mice, and had no effect in DBA/2J mice. On testing the ability to extrapolate the direction of movement of a stimulus, administration of Noopept increased the proportion of correct responses in C57BL/6J and BALB/c mice, but had no effect in DBA/2J mice. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 57, No. 6, pp. 721–728, November–December, 2007.     

Immunological status of nude mice engrafted with allogeneic or syngeneic thymuses

Restoration of T-cell functions and changes in autoantibody production were studied in BALB/c nu/nu (nude) mice engrafted with syngeneic (BALB/c) or allogeneic (C57BL/6J) thymuses across major histocompatability barriers. T-cell functions, including mitogen responses and antibody production to sheep red blood cells (SRBC), were restored in nude mice engrafted with either allogeneic or syngeneic thymuses. Alloreactivity was evaluated by analysis of the pattern of skin allograft rejection, generation of alloreactive cytotoxic T-lymphocytes (CTLs), or quantitation of mixed-lymphocyte reaction (MLR). BABL/c nude mice engrafted with thymuses from newborn C57BL/6J mice accepted the skin from either thymus donor-type mice or from host-type mice. By contrast, such thymic chimeras rejected skin grafts from a third-party donor. CTLs from nude mice engrafted with C57BL/6J thymuses were cytotoxic to target cells of the third party but not to target cells of the host-type or of the thymus-type. In the MLR assay, spleen cells of nude mice engrafted with C57BL/6J thymuses responded vigorously to third party cells and only slightly to cells of the thymus-type. Low levels of serum IgG and high titers of IgM antibodies to nuclear antigens (but not dsDNA) or skin basal cells were also found in nude mice. Antibodies to both nuclear antigens and skin basal cells disappeared after transplantation of syngeneic thymuses, but not after transplantation of allogeneic thymuses. By contrast, serum IgG levels were restored to normal in nude mice engrafted with either syngeneic or allogeneic thymuses. These results suggest that either HLA-matched or HLA-mismatched thymus grafts may become a viable treatment for certain patients with T cell deficiencies associated with deficient development or maintenance of thymic structure and/or function.     

Experimental Cryptosporidium parvum infections in immunosuppressed adult mice.

Five strains of adult mice were immunosuppressed with the synthetic glucocorticosteroid dexamethasone (DEX), administered either orally or intraperitoneally. The strains of mice used were C57BL/6N, DBA/2N, CBA, C3H/HeN, and BALB/cAnN. All mice were evaluated for susceptibility to Cryptosporidium parvum after intragastric inoculation with 10(6) oocysts per mouse. The DBA/2N, CBA, C3H/HeN, and BALB/cAnN mice given 0.25 micrograms of DEX per g per day orally (the dose and route previously used to infect rats with C. parvum) failed to develop chronic infections. However, the C57BL/6N mice sustained light infections during the entire 28-day experiment. The five strains of mice were also administered DEX intraperitoneally at concentrations ranging from 62.5 to 500 micrograms/day. Only the C57BL/6N mice given DEX at 125 micrograms/day developed chronic infections which persisted over 10 weeks, suggesting that the genetic background of the mouse plays a role in determining susceptibility to cryptosporidosis following immunosuppression with DEX. We believe that the C57BL/6N mouse model will prove to be superior to other animal models for evaluating potential anticryptosporidial agents, as well as for elucidating the immunological defects that allow C. parvum to establish chronic infections, because of cost effectiveness and ease in maintenance, breeding, and handling. We also evaluated the C3H/HeJ/beige mouse (lacks natural killer cell activity) and the C57BL/6N mouse maintained on a low-protein diet to induce immunosuppression. Neither of these mice exhibited heavy cryptosporidial infections.     

Experimental Yersinia enterocolitica infection in euthymic and T-cell-deficient athymic nude C57BL/6 mice: comparison of time course, histomorphology, and immune response.

To elucidate the role of T lymphocytes in primary infection with Yersinia enterocolitica, we investigated the elimination rate of this pathogen, the histomorphology of tissue lesions, and the immune responses of athymic T-cell-deficient C57BL/6 nude mice and their euthymic littermates after parenteral infection with Y. enterocolitica of serotype O:8. While a low inoculum of 3 x 10(2) Y. enterocolitica cells (about 0.01 times the median lethal dose for normal C57BL/6 mice) was cleared by normal C57BL/6 mice within 7 to 10 days, athymic nude C57BL/6 mice developed progressive infections after this inoculum, leading to death on days 20 to 25 postinfection (p.i.). While normal C57BL/6 mice experienced short-term transient infections, nude mice exhibited a biphasic, progressive infectious process. Thus, in the early phase (days 1 to 7 p.i.), a rapid influx of CD11b/18-positive cells (Mac-1 antigen) and natural killer cells was evident in the spleens and livers of the nude mice. The late phase (from day 8 p.i. onward) was characterized by a rapid progression of the infection and a further influx of CD11b/18-positive cells into the liver accompanied by an increase in bacterial counts and development of tissue lesions particularly in the liver and spleen. In normal mice, granuloma-like lesions composed of CD11b/18-, CD4-, and CD8-positive cells could be observed. However, granulomata were not found in nude mice. Yersinia-specific immunoglobulin G antibodies appeared on day 15 p.i. in the sera of normal mice, while nude mice failed to develop significant antibody titers. Adoptive transfer of Yersinia-specific T cells into athymic nude mice mediated resistance to Y. enterocolitica infection and restored both the ability of granuloma formation and the production of specific antibodies. In summary, the data presented herein strongly suggest that T lymphocytes play an essential role in the defense of C57BL/6 mice against Y. enterocolitica.     

Serum antibody and ocular responses to murine corneal infection caused by Pseudomonas aeruginosa.

The serum antibody response and differential corneal response to primary and secondary infections by Pseudomonas aeruginosa were investigated in DBA/2J (resistant) and C57BL/6J (susceptible) mice, since they respond differently to intracorneal challenge. Using an enzyme-linked immunosorbent assay, we found that naturally resistant DBA/2J mice mounted a significant immunoglobulin M (IgM) and IgG response to P. aeruginosa within 7 days postinfection of one eye; this was subsequently followed by a drop in the IgM response. Of 31 mice, 30 were able to restore corneal clarity within 3 to 4 weeks. However, when C57BL/6J mice were infected intracorneally, their levels of serum antibody developed more slowly than did those of the DBA/2J mice, and they were unable to restore corneal clarity within 8 to 12 weeks. None of the mice from either test strain mounted a detectable serum IgA response to P. aeruginosa over a 90-day holding period. However, infection of the contralateral, normal cornea of mice of both test strains resulted in a heightened IgG response to P. aeruginosa within 30 days after the secondary infection. Many (50%) of the susceptible C57BL/6J mice recovered or exhibited less severe corneal damage within the 30-day holding period. If the C57BL/6J mice were reinfected 60 days after the primary infection instead of after 30 days, most (89%) of the mice had restored corneal clarity within 3 to 6 days. Passive transfer of immune serum from either recovered DBA/2J or C57BL/6J mice to naive C57BL/6J mice resulted in the restoration of corneal clarity in many of the recipients following infection.     

Nocardia infections in congenitally athymic (nude) mice and in other inbred mouse strains.

The mortality rate and histopathological features of Nocardia asteroides and Nocardia brasiliensis infections in congenitally athymic (nude) mice of ICR and C3H/eB origins were quite different from what we found for Swiss white mice and other inbred mouse strains (namely, C57/BL/6J, New Zealand Black, BALB/c, CBA/LAC, and C3H/eB). The immunocompetent littermates of the congenitally athymic mice occupied an intermediate position between their athymic siblings and Swiss white mice in terms of their responses to both these organisms. Macrophage ingestion and destruction of N. brasiliensis, as demonstrated by electron microscopy, was found to occur. The T-lymphocyte appears to be an essential component in normal mouse resistance to infection by both N. asteroides and N. brasiliensis.     

Distress vocalizations of young in three inbred strains of mice

Vocalizations of young mice of 3 inbred strains, C57BL/6J, BALB/cJ, and C3H/HeJ, were recorded at 3, 6, 9, 12, 15, and 18 days of age. Recordings were made at ambient room temperature (23.5° ± 1.5°C) or under conditions of reduced temperature (2° ± 2°C). Strains differed in terms of rate of signaling, peak frequencies of signals, and average signal duration. Peak frequencies appear to be genetically coded, and did not vary with age or ambient temperature. The effects of reduced ambient temperature on C3H/HeJ and BALB/cJ pups were to extend the age span during which frequent signaling occurs, and to increase the average signal duration throughout that age span. The C57BL/6J pups emitted fewer signals under reduced temperature, but also showed an increase in signal duration. Strain differences in age functions of signaling and the differential impact of experience on the various parameters of the vocalizations suggest that these vocalizations may be a useful behavior in monitoring genetic-experiental interactions and developmental rates in young mice.     

Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was sufficient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100% of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 10(6) sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P. falciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 +/- 260, 88 +/- 38, and 6 +/- 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and < 1% in C3H/HeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.