Roselyne Mancassola

Analysis of Chicken Mucosal Immune Response to Eimeria tenella and Eimeria maxima Infection by Quantitative Reverse Transcription-PCR

ABSTRACT 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. tenellainfection, expression of the proinflammatory cytokine interleukin-1β (IL-1β) mRNA was increased 80-fold. Among the chemokines analyzed, the CC chemokines K203 (200-fold) and macrophage inflammatory factor 1β (MIP-1β) (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-γ) (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-1β and MGF. For these two cytokines, no significant change in expression levels was observed after E. maximainfection. CD3+ intraepithelial lymphocytes may participate in the IFN-γ upregulation observed after infection, since both recruitment and upregulation of the IFN-γ mRNA level were observed in the infected jejunal mucosa. Moreover, in the chicken macrophage cell line HD-11, CC chemokines, MGF, IL-1β, and iNOS were inducible by IFN-γ, suggesting that macrophages may be one of the cell populations involved in the upregulation of these cytokines observed in vivo during infection with Eimeria.

Role of Cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in France

Abstract 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 24h 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 pathogenic agents 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 concomitant peak 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.

Role of Gamma Interferon in Chemokine Expression in the Ileum of Mice and in a Murine Intestinal Epithelial Cell Line after Cryptosporidium parvum Infection

ABSTRACT Cryptosporidium parvum is a protozoan parasite that infects intestinal epithelial cells and induces inflammation of the intestine. To better understand the inflammatory process occurring during cryptosporidiosis, we investigated in this study the kinetics of chemokine expression in the mucosa of mice by quantitative reverse transcription-PCR. Our results demonstrate that among the chemokine mRNAs studied, gamma interferon (IFN-γ)-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), i-TAC, lymphotactin, macrophage inflammatory protein 1β (MIP-1β), and RANTES mRNAs were strongly up-regulated in infected neonate mice, which correlated with the immunofluorescence staining results showing T-cell and macrophage infiltration in the mucosa. Our in vitro data showed that intestinal epithelial cells infected by C. parvum or stimulated by the proinflammatory cytokines (IFN-γ, interleukin-1β, and tumor necrosis factor alpha) produce a pattern of chemokine secretion similar to that observed in vivo, suggesting that these cells may take part in the initial production of chemokines. In order to identify the chemokines responsible for the recruitment of the inflammatory cells leading to a protective immune response, we compared the patterns of chemokine expression in a healing neonate mouse model and a nonhealing IFN-γ knockout (GKO) mouse model of cryptosporidiosis. In the absence of IFN-γ, the chemokine response was altered for IP-10, MIG, i-TAC, RANTES, and MIP-1β mRNAs, while the three ELR C-X-C chemokine mRNAs studied (lipopolysaccharide-induced C-X-C chemokine, MIP-2α, 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.

Cryptosporidium parvum-Specific Mucosal Immune Response in C57BL/6 Neonatal and Gamma Interferon-Deficient Mice: Role of Tumor Necrosis Factor Alpha in Protection

ABSTRACT Both neonatal and C57BL/6 gamma interferon (IFN-γ) 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-γ leads to a protective immunity is yet poorly understood. In order to investigate the effect of IFN-γ 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-γ 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-γ, the adult knockout mice up-regulated the mRNA levels of inflammatory cytokines, such as IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor, in the mucosa, but not tumor necrosis factor alpha (TNF-α), whereas all these cytokines were up-regulated in the infected neonatal mice. Further experiments indicated that injections of TNF-α 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-α may participate in the control of parasite development.

The effect of halofuginone lactate on experimental Cryptosporidium parvum infections in calves

Abstract 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 × 106 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 μg 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 μg 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 μg 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.

Oral and intraperitoneal administration of phosphorothioate oligodeoxynucleotides leads to control of Cryptosporidium parvum infection in neonatal mice.

BACKGROUND Neonates are particularly vulnerable to infections, in part because of the incomplete development of their immune system. Recent advances in immunostimulatory treatments based on conserved microbial components led us to assess the potential of oligodeoxynucleotides (ODNs) for decreasing the sensitivity of neonates to Cryptosporidium parvum infection. METHODS Neonate mice were treated orally or intraperitoneally (ip) with CpG ODNs or non-CpG ODNs 24 h before C. parvum infection, and parasite load and cytokine up-regulation were evaluated. RESULTS CpG ODN 1668 and non-CpG ODN 1668 administered orally, as well as CpG ODN 1668 administered ip, induced an 80%-95% decrease in intestinal parasite load 6 days after infection. Intraperitoneal and oral pretreatment with CpG ODN 1668 led to a strong initial up-regulation of cytokines and CD69 messenger RNA in the intestine and a decrease in parasite load by a Toll-like receptor 9 (TLR9)-dependent mechanism. By contrast, oral administration of non-CpG ODN 1668 decreased parasite load by a TLR9-independent mechanism. CONCLUSION 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.

EVALUATION OF DECOQUINATE TO TREAT EXPERIMENTAL CRYPTOSPORIDIOSIS IN KIDS

The purpose of this trial was to evaluate the effects of decoquinate at 2.5 mg/kg/day for 21 days to prevent an experimental cryptosporidiosis in kids. Twenty 1-day-old male kids (French Alpin), fed initially goat colostrum heated 1 h at 56 degrees C and fed twice daily with nonmedicated milk replacer, were assigned into 2 groups. Kids of both groups were orally inoculated with 10(6) Cryptosporidium parvum (D0 = inoculation day). Group A kids were kept as nonmedicated controls and group B kids were orally medicated with 2.5 mg/kg/day of decoquinate (Deccox L. Rhône Poulenc Animal Nutrition) for 21 days from D-3 to D17. The studied criteria were body weight gain, oocyst shedding and specific anti-C. parvum immune response. In group A, the inoculation was not followed by mortality; but only by diarrhea and high oocyst shedding. Decoquinate reduced the severity of cryptosporidiosis in group B kids. The treatment prevented episodes of diarrhea and weight gain decrease for the D0-D7 and D0-D14 disease periods but did not allow a better final weight gain. The oocyst shedding was decreased in number and in duration. This parasitic development has induced a specific anti-C. parvum immune response. This drug is well-tolerated by animals and may be recommended in the prevention of ruminant cryptosporidiosis, a disease which has very limited treatment options.

Treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum

Ovine or bovine colostrums with different antibody titers were tested for their ability to prevent cryptosporidiosis in five groups of neonatal lambs experimentally infected with 10(6) Cryptosporidium parvum oocysts 2 days after birth (Day 0). In a control group (Group 1), six lambs were deprived of ewe colostrum and exclusively fed with milk replacer. Two groups of six lambs were allowed to suckle their non-hyperimmunized (Group 2) or hyperimmunized (Group 3) dams throughout the experiment. Two groups of seven lambs were separated from their dams at birth before suckling and fed with non-hyperimmune (Group 4) or hyperimmune (Group 5) bovine colostrum; for 7 days they received 50 ml of colostrum completed by milk replacer twice a day, then they were fed with milk replacer exclusively. Control lambs became infected and developed clinical cryptosporidiosis with diarrhea on Days 4-9 post inoculation, oocyst shedding and mortality (2/6). In all the treated groups, the colostrum prevented mortality and clinical cryptosporidiosis. The mortality (5/7) observed in Group 5 was not due to cryptosporidiosis but anemia. In treated groups, specific antibodies were detected on Day 0 after 2 days of colostrum intake and varied little in time for IgM and IgG in spite of the parasite development, whereas they appeared later in the control group, on Day 4 for IgM, Day 11 for IgA and Day 14 for IgG. In all groups, the response which was the most consistent was the IgA response which evolved from Days 11 to 18 in association with the decline of oocyst shedding. Our results show that whatever the serum antibody titers were, the specific C. parvum circulating antibodies have no influence on the control of cryptosporidiosis. The prophylaxis or the treatment of cryptosporidiosis require high titers of specific C. parvum antibodies in the gut lumen during a sufficiently long period.

Neonate Intestinal Immune Response to CpG Oligodeoxynucleotide Stimulation

Background The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates. Methodology/Principal Findings First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice. Conclusions/Significance This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases.

Analysis of humoral immune response in chickens after inoculation with Cryptosporidium baileyi or Cryptosporidium parvum.

White leghorn chickens aged 14 days were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium baileyi or C. parvum to compare the specific immune responses. Cross-reactions were evaluated using homologous or heterologous antigens in enzyme-linked immunosorbent assay (ELISA) and Western blot to determine the occurrence of an antigenic homology between these two species. Blood, bile, whole intestine, bursa of Fabricius, and feces were collected daily from the day of inoculation (day 0) to day 22 postinoculation (PI). Eight control chickens remained negative up to day 22 PI. Chickens inoculated with C. baileyi did not express clinical symptoms but shed oocysts from days 6 to 21 PI. Chickens inoculated with C. parvum exhibited no clinical signs, no oocysts in feces, and no developmental stages of the parasite. However, a specific immune response to both antigens appeared on day 9 PI. ELISA using homologous or heterologous antigens showed that anti-C. baileyi and anti-C. parvum antibodies in serum or bile were detectable using C. baileyi or C. parvum oocysts as antigen, but the intensity of the response was significantly higher when C. baileyi was used. Cross-reactions in immunoblot analysis confirmed ELISA results, revealing a greater number of bands using C. baileyi as antigen but showing that epitopes recognized on the protein with a molecular weight of 15,000-17,000 were different.