Both neonatal and C57BL/6 gamma interferon (IFN-␥) knockout (C57BL/6-GKO) mice are susceptible to Cryptosporidium parvum, but the course of infection is different. Neonatal mice are able to clear the parasite within 3 weeks, whereas C57BL/6-GKO mice, depending on age, die rapidly or remain chronically infected. The mechanism by which IFN-␥ leads to a protective immunity is yet poorly understood. In order to investigate the effect of IFN-␥ on other cytokines expressed in the intestinal mucosa during C. parvum infection, we studied cytokine mRNA expression in the neonates and GKO (neonatal and adult) mice by quantitative reverse transcription-PCR (RT-PCR) at 4 and 9 days after infection. IFN-␥ mRNA levels were quickly and strongly up-regulated in the mucosa of neonatal mice. In GKO mice, the Th1-type response was dramatically altered during the infection, whereas the mRNA expression levels of the Th2-type cytokines interleukin 4 (IL-4) and IL-10 were increased in both mouse models. In the absence of IFN-␥, the adult knockout mice up-regulated the mRNA levels of inflammatory cytokines, such as IL-1␤, IL-6, and granulocyte-macrophage colony-stimulating factor, in the mucosa, but not tumor necrosis factor alpha (TNF-␣), whereas all these cytokines were upregulated in the infected neonatal mice. Further experiments indicated that injections of TNF-␣ into GKO adult mice significantly reduced oocyst shedding. The results of the present study indicate that the resolution of infection is dependent on the expression of Th1-type cytokines in the mucosa of C57BL/6 mice and that TNF-␣ may participate in the control of parasite development.Cryptosporidium parvum is an obligate intracellular protozoan parasite that infects intestinal epithelial cells of humans and various other mammals. C. parvum causes protracted diarrhea in young and immunodeficient individuals and can lead to death for AIDS patients. Cryptosporidiosis is frequent in young farm animals and has economic and environmental consequences. In immunocompetent hosts, the disease is self-limited, suggesting a major role for host defense factors in controlling the infection.Most of the studies of experimental cryptosporidiosis have been performed with rodents whose immune systems were impaired, e.g., neonatal mice (14,25,35), rats immunosuppressed with dexamethasone (27), or congenitally mutated nude (21, 23) and SCID mice (17,34). More recent studies have used mice with targeted mutations for the genes of major histocompatibility complex class II (1), CD40, CD40L (7), or gamma interferon (IFN-␥) (33, 38). The key role of IFN-␥ in resistance to C. parvum infection initially demonstrated with antibody depletion was confirmed more recently with IFN-␥ knockout mice (GKO) (6,33,34). However, the mechanisms whereby IFN-␥ intervenes in the clearance of C. parvum are still not well understood. Some possibilities, not mutually exclusive, include a direct toxic effect of IFN-␥ on the parasite or the infected cells or the induction of other cytokines that can be toxic for the pa...

The recent cloning of chicken genes coding for interleukins, chemokines, and other proteins involved in immune regulation and inflammation allowed us to analyze their expression during infection with Eimeria. The expression levels of different genes in jejunal and cecal RNA extracts isolated from uninfected chickens and chickens infected with Eimeria maxima or E. tenella were measured using a precise quantitative reverse transcription-PCR technique. Seven days after E. tenella infection, expression of the proinflammatory cytokine interleukin-1␤ (IL-1␤) mRNA was increased 80-fold. Among the chemokines analyzed, the CC chemokines K203 (200-fold) and macrophage inflammatory factor 1␤ (MIP-1␤) (80-fold) were strongly upregulated in the infected ceca, but the CXC chemokines IL-8 and K60 were not. However, the CXC chemokines were expressed at very high levels in uninfected cecal extracts. The levels of gamma interferon (IFN-␥) (300-fold), inducible nitric oxide synthase (iNOS) (200-fold), and myelomonocytic growth factor (MGF) (50-fold) were also highly upregulated during infection with E. tenella, whereas cyclooxygenase 2 showed a more modest (13-fold) increase. The genes upregulated during E. tenella infection were generally also upregulated during E. maxima infection but at a lower magnitude except for those encoding MIP-1␤ and MGF. For these two cytokines, no significant change in expression levels was observed after E. maxima infection. CD3؉ intraepithelial lymphocytes may participate in the IFN-␥ upregulation observed after infection, since both recruitment and upregulation of the IFN-␥ mRNA level were observed in the infected jejunal mucosa. Moreover, in the chicken macrophage cell line HD-11, CC chemokines, MGF, IL-1␤, and iNOS were inducible by IFN-␥, suggesting that macrophages may be one of the cell populations involved in the upregulation of these cytokines observed in vivo during infection with Eimeria.Chicken coccidiosis is caused by intracellular protozoan parasites belonging to seven species of Eimeria. These parasites invade and reside in the lining of the intestine or ceca. Parasite development causes diarrhea, morbidity, and mortality, and the impact of coccidiosis on the industry has serious economic consequences. Thus far, chemoprophylaxis has controlled the disease but has been complicated by the emergence of drug resistance. Infection by Eimeria promotes antibody and cellmediated immune responses. However, cellular immunity mediated by various cell populations, including T lymphocytes, NK cells, and macrophages, plays a major role in disease resistance (27). There is increasing evidence of CD4 ϩ and intraepithelial lymphocyte (IEL) involvement during a primary infection, while T-cell receptor ␣-and ␤-chain-positive CD8 ϩ IEL play a key role in secondary infection (25). The development of a vaccine has been hampered by the lack of understanding of the various components of the host immune system involved in protective immunity.The low level of homology between chicken genes and their mammalian co...

Cryptosporidium parvum is a protozoan parasite that infects intestinal epithelial cells and induces inflam-In the absence of IFN-␥, the chemokine response was altered for IP-10, MIG, i-TAC, RANTES, and MIP-1␤ mRNAs, while the three ELR C-X-C chemokine mRNAs studied (lipopolysaccharide-induced C-X-C chemokine, MIP-2␣, and KC mRNAs) were strongly overexpressed. These results are consistent with the neutrophil recruitment observed in the lamina propria of GKO mice at day 9 postinfection but are not consistent with the hypothesis that these cells play an important role in the resolution of the infection. On the contrary, the altered response of chemokines responsible for the recruitment of macrophages and T cells in GKO mice suggests that these two populations may be critical in the development of a protective immune response.

This study was carried out to find the importance of Cryptosporidium parvum in diarrhoea of neonatal calves in two types of breeding - suckling and dairy calves - in France. Different agents causing neonatal diarrhoea, E. coli, rotavirus, coronavirus, Salmonella and Cryptosporidium were systematically researched in faeces. 1. Suckling calves: In 40 livestock farms selected for diarrhoea, 311 calves 4 to 10 days old which had diarrhoea for less than 24h or no diarrhoea, were included in the study. A prophylaxis of neonatal diarrhoea had been carried out in 21 of the 40 livestock farms. On D0 (inclusion day), the mean age was 6 days, 82% presented a good initial general condition and 76.2% had a good appetite; 48.6% were diarrhoeic but 91.3% presented no sign of dehydration. Only 6.1% were infected by E. coli K99, 14.3% by rotavirus, 6.8% by coronavirus, 0.3% by Salmonella but 50% excreted C. parvum oocysts. This later percentage increases up to 84% and 86% by D3 and D7, respectively . We note that 16% of the 4-day-old calves on D0 are excreting oocysts and this percentage increases as a function of the age of the calf on D0 to reach 90% to 95% by the age of 8 days. 10 out of 12 dead calves excreted C. parvum oocysts. From D0 to D14 the other pathogen agents show a relative or a decreasing stability. 2. Dairy calves: 382 calves which had diarrhoea for less than 24 h or no diarrhoea, aged 8 to 15 days coming from six industrial livestock farms were included in the study. On D0, 99% of the calves presented a good initial general condition, 99.7% had a good appetite and no calf was dehydrated. At this date (D0), 16.8% of the calves excreted cryptosporidia. This percentage increases up to 23% and 51.8% on D3 and D8, respectively, then decreases to 31.9% on D14. The pressure of the other pathogenicagents remains relatively stable, excepted for rotavirus on D7 (from 9.9% on D0 to 27.2% on D7, then 12.6% on D14) which does not explain the concomitantpeak in diarrhoea because the infection by rotavirus on D7 is more frequent in non-diarrhoeic calves than in diarrhoeic calves. Our results show that Cryptosporidium prevalence is higher in suckling than in dairy calves and C. parvum constitutes actually in both cases the major aetiological agent of neonatal diarrhoea.

The chemoprophylactic effects of halofuginone lactate were tested against calf experimental cryptosporidiosis. Twenty 2-day-old calves, divided into four groups, were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium parvum. The infected control group was unmedicated whereas the three other groups were medicated with the drug at 30, 60 and 120 micrograms kg-1 day-1, respectively, for 7 days, from Day (D) 2 to D8 post-inoculation (D 0 was inoculation day). The calves were weighed twice weekly and disease development and drug efficacy were assessed daily from D0 to D30 from consistency of feces, shedding of oocysts and mortality. Experimental C. parvum infection caused a severe clinical disease with profuse watery diarrhea, high oocyst shedding and mortality (3 out of 5) in the unmedicated group. The results clearly demonstrated the efficacy of halofuginone lactate in reducing the severity of clinical cryptosporidiosis. This efficacy was dose-dependent. The lowest dose (30 micrograms kg-1 day-1) was not able to prevent clinical disease and mortality (3 out of 5). No clinical signs were observed with the 60 and 120 micrograms kg-1 day-1 doses, but the animals shed oocysts after drug withdrawal. This shedding was more delayed the higher the dose of drug administered, but the delayed shedding had no effect on the growth of the animals.

The control of neonatal C. parvum infection by ip or oral administration of ODNs is feasible by 2 different mechanisms: (1) the well-known interaction involving CpG/TLR9, leading to the production of cytokines and lymphocyte activation, and (2) a new unknown mechanism that is independent of TLR9 and effective orally.

The neonatal intestinal immune system is still undergoing development at birth, leading to a higher susceptibility to mucosal infections. In this study, we investigated the effect of poly(I:C) on controlling enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. After poly(I:C) administration, a rapid reduction in parasite burden was observed and proved to be dependent on CD11c(+) cells and TLR3/TRIF signaling. Protection against C. parvum required additional signals provided by the gut flora through TLR5 and MyD88 signaling. This cooperation gave rise to higher levels of expression of critical mutually dependent cytokines such as interleukin 12p40 and type 1 and type 2 interferons, the last 2 being known to play a key role in the elimination of infected enterocytes. Our findings demonstrate in neonatal mice how gut flora synergizes with poly(I:C) to elicit protective intestinal immunity against an intracellular pathogen.

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