Erik P. Lillehoj

Avian Coccidiosis. A Review of Acquired Intestinal Immunity and Vaccination Strategies

The gut-associated lymphoid tissues contain B and T lymphocytes responsible for acquired immunity to avian coccidiosis. Intestinal B cells begin producing parasite-specific antibodies shortly after infection although their role in protecting against coccidiosis is debated. T-cell-mediated immunity, predominantly by intestinal intraepithelial lymphocytes and lamina propria lymphocytes, confers the main component of protective immunity to Eimeria. Many of these cells display the CD8 and gammadelta T-cell receptor surface antigens, phenotypic markers of cytotoxic T cells. Although their role in eliminating Eimeria infection remains to be completely elucidated, T cells have been implicated in parasite transport, and their activity is augmented by interferon-gamma and interleukin-2. Because of the importance of cell-mediated immunity, coccidiosis vaccines must be capable of stimulating intestinal T cells. Orally delivered, live parasite vaccines, either unattenuated or attenuated, are powerful stimulators of intestinal cell-mediated immunity, but antigenic variability between Eimeria species present in the vaccine and in the field may restrict their commercial application. The newer generations of recombinant DNA and subunit protein vaccines, particularly when used in conjunction with interferon-gamma and interleukin-2, have shown preliminary promise in controlling experimental infections but have yet to be commercially developed.

Intestinal immune responses to coccidiosis

Intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and production efficiency of livestock and poultry. Coccidiosis is an intestinal infection caused by intracellular protozoan parasites belonging to several different species of Eimeria. Infection with coccidia parasites seriously impairs the growth and feed utilization of chickens and costs the US poultry industry more than

Antibodies to Toxoplasma gondii in Individuals with First-Episode Schizophrenia

1.5 billion in annual losses. Although acquired immunity to Eimeria develops following natural infection, due to the complex life cycle and intricate host immune response to Eimeria, vaccine development has been difficult and a better understanding of the basic immunobiology of pertinent host-parasite interactions is necessary for developing effective immunological control strategies against coccidiosis. Chickens infected with Eimeria produce parasite specific antibodies in both the circulation and mucosal secretions but humoral immunity plays only a minor role in protection against this disease. Rather, recent evidence implicates cell-mediated immunity as the major factor conferring resistance to coccidiosis. This review will summarize current understanding of the avian intestinal immune system and its response to Eimeria as well as provide a conceptual overview of the complex molecular and cellular events involved in intestinal immunity to coccidiosis. It is anticipated that increased knowledge of the interaction between parasites and host immunity will stimulate the birth of novel immunological and molecular biological concepts in the control of intestinal parasitism.

Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens

We employed enzyme immunoassay (EIA) and Western blotting techniques to measure the level of antibodies to Toxoplasma gondii proteins in serum samples from 38 individuals undergoing their first episode of schizophrenia and from a group of matched control subjects. We found that the individuals with first-episode schizophrenia had significantly increased levels of IgG, IgM, and IgA class antibodies to Toxoplasma proteins, as compared with the control subjects.

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice.

This study was conducted to compare growth performance, gut morphometry, and parameters of local and systemic immunity in broiler chickens fed for 22 consecutive days with a diet supplemented with Bacillus spp. as direct-fed microbials (DFM), a commercial product incorporating 3 DFM, or a nonsupplemented diet. Direct-fed microbials did not significantly modify BW gain and most failed to affect serum antibody levels in response to immunization with a recombinant Eimeria protein. However, altered intestinal morphometric measurements were readily apparent in DFM-fed chickens as revealed by increased villus height and crypt depth compared with non-DFM-fed controls. In addition, serum levels of alpha-1-acid glycoprotein as an inflammatory marker were reduced in DFM-fed birds, whereas splenic lymphocyte proliferation, intestine intraepithelial lymphocyte subpopulations, and cytokine mRNA levels in intraepithelial lymphocytes were increased, decreased, or unchanged compared with controls depending on the DFM used. These results provide a rational scientific basis for future studies to investigate DFM as immunomodulating agents to enhance host protective immunity against enteric pathogens in broiler chickens.

A recombinant Eimeria protein inducing interferon-γ production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis.

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1−/− mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1−/− mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-α and KC in bronchoalveolar lavage fluid, higher levels of TNF-α in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.

Protective Immunity against Eimeria acervulina following In Ovo Immunization with a Recombinant Subunit Vaccine and Cytokine Genes

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Immunopathology and Cytokine Responses in Broiler Chickens Coinfected with Eimeria maxima and Clostridium perfringens with the Use of an Animal Model of Necrotic Enteritis

ABSTRACT A purified recombinant protein from Eimeria acervulina (3-1E) was used to vaccinate chickens in ovo against coccidiosis both alone and in combination with expression plasmids encoding the interleukin 1 (IL-1), IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or gamma interferon (IFN-γ) gene. When used alone, vaccination with 100 or 500 μg of 3-1E resulted in significantly decreased oocyst shedding compared with that in nonvaccinated chickens. Simultaneous vaccination of the 3-1E protein with the IL-1, -15, -16, or -17 gene induced higher serum antibody responses than 3-1E alone. To evaluate protective intestinal immunity, vaccinated birds were challenged with live E. acervulina oocysts 14 days posthatch, and fecal-oocyst shedding and body weight gain were determined as parameters of coccidiosis. Chickens vaccinated with 3-1E protein showed significantly lower oocyst shedding and normal body weight gain than nonvaccinated and infected controls. Simultaneous immunization with 3-1E and the IL-2, -15, -17, or -18 or IFN-γ gene further reduced oocyst shedding compared with that achieved with 3-1E alone. These results provide the first evidence that in ovo vaccination with the recombinant 3-1E Eimeria protein induces protective intestinal immunity against coccidiosis, and this effect was enhanced by coadministration of genes encoding immunity-related cytokines.

Resistance to Intestinal Coccidiosis Following DNA Immunization with the Cloned 3-1E Eimeria Gene Plus IL-2, IL-15, and IFN-γ

Abstract The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-α, IFN-γ, IL-1β, IL-2, IL-12, IL-13, IL-17, and TGF-β4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens.

Airway mucus: its components and function

Abstract A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-γ. Vaccination efficacy was assessed by increased serum anti–3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti–3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (≥25 μg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-γ genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-γ significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.