Muriel Naciri

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.

Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA

In this study the effectiveness of a DNA vaccine to confer protection against cryptosporidiosis, an enteric infection of lifestock and humans, was evaluated. A vaccination protocol using a recombinant plasmid encoding the 15 kDa surface sporozoite protein of Cryptosporidium parvum was developed in adult pregnant goats. The present study reports that nasal immunization of pregnant goats with CP15-DNA led to a transfer of immunity to offspring conferring protection against C. parvum infection. Kids from CP15-DNA-vaccinated dams shed significantly fewer oocysts and over a shorter period than did kids from unvaccinated goats. The low level of parasite development in protected kids did not affect their growth whereas unprotected kids grew much slowly. There was still a significant difference in the weights of protected and unprotected kids after complete recovery. Anti-CP15 antibodies were present in serum and colostrum from vaccinated goats. Nevertheless, the precise immune mechanism of protection has still to be determined. This vaccine should reduce the economic losses due to cryptosporidiosis in ruminants, specially in small ruminants (calves, lambs, kids). It has also the potential to reduce environmental contamination by reducing oocyst shedding.

Prevalence of Cryptosporidium infection in calves in France.

Abstract Two multicentre surveys were conducted in France to estimate the prevalence of Cryptosporidium infection in calves using qualitative ELISA for detection of Cryptosporidium coproantigens and oocysts. The first survey involved 4–12-day-old calves in six dairy-calf distribution centres, collecting calves from seven Administrative Regions (Aquitaine, Bretagne, Franche-Comté, Lorraine, Normandie, Nord, Pays de Loire). For each region, 20 calves were selected every month for 12 consecutive months (October 1995–September 1996). Prevalence of Cryptosporidium infection was 17.9% (Confidence Intervals (C.I.) 95%=[16.1%; 19.8%]) among the 1628 selected calves, of which only 5.3% had diarrhoea. The second survey conducted between November 1995 and May 1996 involved 4–21-day-old calves examined by veterinary practitioners who selected 189 livestock farms of dairy- or suckler-type in ten Administrative Departments (Allier, Cantal, Creuse, Doubs, Ille-et-Vilaine, Maine-et-Loire, Manche, Pas-de-Calais, Saône-et-Loire, Vendée). Cryptosporidia were detected in 105 (55.6%) of the farms. Among the 440 calves examined, of which 398 (90.5%) presented diarrhoea, cryptosporidia were found in 191 animals, i.e. a prevalence of 43.4% (C.I. 95%=[38.8%; 48.0%]). Breed of calves and type of housing had very little impact on prevalence in this survey. Some regional variations could be noticed, even if cryptosporidia infection is widespread. Monthly variations could be related to seasonal peaks in calving with a lower infection rate during summer.

Major antigens of Cryptosporidium parvum recognised by serum antibodies from different infected animal species and man

Serum humoral immune response to Cryptosporidium parvum was evaluated in six species: mouse, rabbit, lamb, calf, pig and man. Electrophoretic and immunoblot analysis showed that specific animal antibody response appeared between Day 4 and Day 15 post inoculation. The two main target antigens had apparent molecular weights of 15-17 and 23 kDa. They were recognised by each species studied. Serum IgA intensively recognised the 15-17 kDa antigen, except in rabbit. This study demonstrates that these two antigens are consistent targets of humoral immune response and can therefore be of great interest in studies of therapy/prophylaxis.

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.