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...

This protocol is part of the DNA workflow applied in the Eco-ALpsWater Project, here in particular to characterize the diversity of diatom assemblage in biofilms or plankton samples. Different studies have already revealed the potential of diatom metabarcoding applications for freshwater quality assessment (Kermarrec et al. 2014; Vasselon et al. 2017ab; Visco et al. 2015). The choice of the marker gene and barcode region is key for obtaining relevant inventories of diversity and precise taxonomic assignment. For benthic diatoms, the rbcL gene has proved to be an appropriate taxonomic marker for biomonitoring (Kermarrec et al. 2013, 2014; Vasselon et al. 2017a,b) and a well-curated barcode reference library is already available to assign species names to rbcL sequences (R-Syst::diatom, Rimet et al. 2016). For the Eco-AlpsWater project, biolfilms sampled in rivers and lakeshores are collected as described in the dedicated protocols ("Lake plankton sample collection ..." and "Biofilms sample collection ...") and DNA is extracted as described in the protocol "Biofilms DNA extraction" ; all these protocols are part of the Deliverable D.T1.1.2. We present here the following step in the DNA workflow (i.e. PCR amplification of selected barcodes, and wet lab methods to prepare DNA library for downstream MiSeq Sequencing). This protocol has been used in recent studies (e.g. Keck et al 2018 ; Vasselon et al 2018) where diatoms metabarcoding has been used for ecological assessment of rivers. Several primers were proposed in the literature to characterize Diatom communities through environmental DNA metabarcoding approaches, including the 18S, COI and rbcL barcodes. Following the recommendation provided by Kermarrec et al. 2014, who compared the efficiency of those 3 barcodes to accurately characterize diatom communities from freshwater samples (lakes and rivers), the rbcL barcode will be used within the Eco-AlpsWater project as he provides a good taxonomical resolution.