Effects of in ovo injection with selenium on immune and antioxidant responses during experimental necrotic enteritis in broiler chickens.

This study was conducted to investigate the effects of in ovo injection of Se on modulating the immune system and antioxidant responses in broiler chickens with experimental necrotic enteritis. Broiler eggs were injected at 18 d of embryo age with either 100 µL of PBS alone or sodium selenite (Na2SeO3) in PBS, providing 0 (SS0), 10 (SS10), or 20 (SS20) µg of Se/egg. At 14 d posthatch, PBS-treated and uninfected chickens were kept as the control group, whereas the remaining chickens were orally infected with 1.0 × 10(4) sporulated oocysts of Eimeria maxima (SS0, SS10, SS20). At 18 d posthatch, E. maxima-infected chickens were orally infected with 1.0 × 10(9) cfu of Clostridium perfringens. Infected control SS0 group showed significantly decreased BW compared with the uninfected control. However, SS20 group showed significantly increased BW compared with the infected control SS0 group, whereas the BW were similar among uninfected control and infected SS10 and SS20 groups. The SS10 group showed significantly lower intestinal lesions compared with the SS0 group, and oocyst production was decreased in both SS10 and SS20 groups. Serum malondialdehyde level and catalase activity were also decreased in both SS10 and SS20 groups, whereas the superoxide dismutase level was significantly lower in the SS10 group compared with the SS0 group. The SS20 group showed significantly higher levels of transcripts for IL-1ß and IL-6 in intestine, and SS10 and SS20 groups had higher levels of transcripts for IL-8 and inducible nitric oxide synthase expression and decreased glutathione peroxidase 7 mRNA levels compared with the SS0 group. The SS10 and SS20 groups also showed increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with the SS0 group. These collective results suggest that the injection of Se into the amniotic cavity of developing eggs may be beneficial for enhancing immune and antioxidant responses in the hatched chickens exposed to the necrotic enteritis-causing pathogens.

Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens.

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

Effect of dietary antimicrobials on immune status in broiler chickens.

This study evaluated the effects of dietary anticoccidial drugs plus antibiotic growth promoters (AGPs) on parameters of immunity in commercial broiler chickens. Day-old chicks were raised on used litter from a farm with endemic gangrenous dermatitis to simulate natural pathogen exposure and provided with diets containing decoquinate (DECX) or monensin (COBN) as anticoccidials plus bacitracin methylene disalicylate and roxarsone as AGPs. As a negative control, the chickens were fed with a non-supplemented diet. Immune parameters examined were concanavalin A (ConA)-stimulated spleen cell proliferation, intestine intraepithelial lymphocyte (IEL) and spleen cell subpopulations, and cytokine/chemokine mRNA levels in IELs and spleen cells. ConA-induced proliferation was decreased at 14 d post-hatch in DECX-treated chickens, and increased at 25 and 43 d in COBN-treated animals, compared with untreated controls. In DECX-treated birds, increased percentages of MHC2(+) and CD4(+) IELS were detected at 14 d, but decreased percentages of these cells were seen at 43 d, compared with untreated controls, while increased TCR2(+) IELs were evident at the latter time. Dietary COBN was associated with decreased fractions of MHC2(+) and CD4(+) IELs and reduced percentages of MHC2(+), BU1(+), and TCR1(+) spleen cells compared with controls. The levels of transcripts for interleukin-4 (IL-4), IL-6, IL-17F, IL-13, CXCLi2, interferon-γ (IFN-γ), and transforming growth factorß4 were elevated in IELs, and those for IL-13, IL-17D, CXCLi2, and IFN-γ were increased in spleen cells, of DECX- and/or COBN-treated chickens compared with untreated controls. By contrast, IL-2 and IL-12 mRNAs in IELs, and IL-4, IL-12, and IL-17F transcripts in spleen cells, were decreased in DECX- and/or COBN-treated chickens compared with controls. These results suggest that DECX or COBN, in combination with bacitracin and roxarsone, modulate the development of the chicken post-hatch immune system.

Impact of fresh or used litter on the posthatch immune system of commercial broilers.

This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.

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

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.

In vitro effects of plant and mushroom extracts on immunological function of chicken lymphocytes and macrophages.

1. The present study was conducted to examine the effects of organic extracts from milk thistle (Silybum marianum), turmeric (Curcuma longa), reishi mushroom (Ganoderma lucidum), and shiitake mushroom (Lentinus edodes) on innate immunity and tumor cell viability. 2. Innate immunity was measured by lymphocyte proliferation and nitric oxide production by macrophages, and the inhibitory effect on tumor cell growth was assessed using a non-radioactive assay. For measuring the cytokine levels in the HD11 macrophages which were treated with extracts of turmeric or shiitake mushroom, the levels of mRNAs for interferon-alpha (IFN- alpha), interleukin-1beta (IL-1beta), IL-6, IL-12, IL-15, IL-18, and tumor necrosis factor superfamily 15 (TNFSF15) were quantified by real time RT-PCR. 3. In vitro culture of chicken spleen lymphocytes with extracts of milk thistle, turmeric, and shiitake and reishi mushrooms induced significantly higher cell proliferation compared with the untreated control cells. Stimulation of macrophages with extracts of milk thistle and shiitake and reishi mushrooms, but not turmeric, resulted in robust nitric oxide production to levels that were similar with those induced by recombinant chicken interferon-gamma. All extracts uniformly inhibited the growth of chicken tumor cells in vitro at the concentration of 6.3 through 100 microg/ml. Finally, the levels of mRNAs encoding IL-1beta, IL-6, IL-12, IL-18, and TNFSF15 were enhanced in macrophages that were treated with extracts of turmeric or shiitake mushroom compared with the untreated control. 4. These results document the immunologically-based enhancement of innate immunity in chickens by extracts of plants and mushrooms with known medicinal properties in vitro. In vivo studies are being planned to delineate the cellular and molecular mechanisms responsible for their mechanism of action.

Association of resistance to avian coccidiosis with single nucleotide polymorphisms in the zyxin gene.

Our previous genetic studies demonstrated that resistance to avian coccidiosis is linked with microsatellite markers LEI0071 and LEI0101 on chromosome 1. In this study, the associations between parameters of resistance to coccidiosis and single nucleotide polymorphisms (SNP) in 3 candidate genes located between LEI0071 and LEI0101 [zyxin, CD4, and tumor necrosis factor receptor super family 1A (TNFRSF1A)] were determined. The SNP were genotyped in 24 F(1) generation and 290 F(2) generation animals. No SNP were identified in the TNFRSF1A gene, whereas 10 were located in the zyxin gene and 4 in the CD4 gene. At various times following experimental infection of the F(2) generation with Eimeria maxima, BW, fecal oocyst shedding, and plasma levels of carotenoid, nitrite plus nitrate (NO(2)(-) + NO(3)(-)), and interferon-gamma (IFN-gamma) were measured as parameters of resistance. Single marker and haplotype-based tests were applied to determine the associations between the 14 SNP and the parameters of coccidiosis resistance. None of the CD4 SNP were correlated with disease resistance. However, by single marker association, several of the zyxin SNP were significantly associated with carotenoid or NO(2)(-) + NO(3)(-) concentrations. These were the SNP at nucleotide 149 associated with carotenoid at d 3 postinfection (PI), nucleotide 187 with carotenoid at d 6 and 9 PI, and nucleotide 159 with carotenoid between d 3 and 9 PI. In addition, the zyxin SNP at nucleotide 191 was significantly associated with increased levels of NO(2)(-) + NO(3)(-) at d 3 PI. By haplotype association, the zyxin SNP also were found to be highly associated with NO(2)(-) + NO(3)(-) at d 3 PI and increased IFN-gamma at d 6 PI. These results suggest that zyxin is a candidate gene potentially associated with increased resistance to experimental avian coccidiosis.

Induction of passive immunity in broiler chickens against Eimeria acervulina by hyperimmune egg yolk immunoglobulin Y.

The protective effect of hyperimmune IgY fraction of egg yolk prepared from hens hyperimmunized with multiple species of Eimeria oocysts on experimental coccidiosis was evaluated in young broilers. Chickens were continuously fed from hatch with a standard diet containing hyperimmune IgY egg yolk powder or a nonsupplemented control diet and orally challenged at d 7 posthatch with 5.0 x 10(3) sporulated Eimeria acervulina oocysts. Body weight gain between d 0 and 10 and fecal oocyst shedding between d 5 and 10 postinfection were determined as parameters of protective immunity. Chickens given 10 or 20% hyperimmune IgY egg yolk powder showed significantly increased BW gain and reduced fecal oocyst shedding compared with control birds fed the nonsupplemented diet. In another trial, lower IgY concentrations (0.01, 0.02, and 0.05%) were used to treat birds with 1.0 x 10(4) oocysts of E. acervulina. Total oocyst shedding was significantly (P < 0.05) reduced in chickens fed the 0.02 and 0.05% hyperimmune IgY supplemented-diets compared with animals fed the nonsupplemented diet. Similarly, chickens fed 0.5% of hyperimmune IgY egg yolk powder diet and challenged with 1.0 x 10(4) oocysts exhibited reduced oocyst shedding compared with the control birds given 0.5% of IgY from nonimmunized hen eggs, although BW gain was not affected. We conclude that passive immunization of chickens with anti-coccidia IgY antibodies provide protective immunity against coccidiosis challenge infection.

Immune-related gene expression in two B-complex disparate genetically inbred Fayoumi chicken lines following Eimeria maxima infection.

To investigate the influence of genetic differences in the MHC on susceptibility to avian coccidiosis, M5.1 and M15.2 B-haplotype-disparate Fayoumi chickens were orally infected with live Eimeria maxima oocysts, and BW gain, fecal oocyst production, and expression of 14 immune-related genes were determined as parameters of protective immunity. Weight loss was reduced and fecal parasite numbers were lower in birds of the M5.1 line compared with M15.2 line birds. Intestinal intraepithelial lymphocytes from M5.1 chickens expressed greater levels of transcripts encoding interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, IL-15, IL-17A, inducible nitric oxide synthase, and lipopolysaccharide-induced tumor necrosis factor-alpha factor and lower levels of mRNA for IFN-alpha, IL-10, IL-17D, NK-lysin, and tumor necrosis factor superfamily 15 compared with the M15.2 line. In the spleen, E. maxima infection was associated with greater expression levels of IFN-gamma, IL-15, and IL-8 and lower levels of IL-6, IL-17D, and IL-12 in M5.1 vs. M15.2 birds. These results suggest that genetic determinants within the chicken MHC influence resistance to E. maxima infection by controlling the local and systemic expression of immune-related cytokine and chemokine genes.

Amino acid sequence analysis of the H-2Kk alloantigen: complete sequence of residues 1-98 and partial sequence from 99 to 263.

The H-2Kk molecule was purified by immunoprecipitation from the glycoprotein fraction of Nonidet P-40 extracts of RDM 4 mouse tumor cells. Cyanogen bromide cleavage of the major papain fragment yielded three peptides, the largest of which consisted of three disulfide-linked peptides which could be separated after reduction and alkylation. These peptides were readily aligned by their homology to similar fragments derived from other H-2 class I molecules. Amino acid sequence analyses of the two nondisulfide-linked peptides, peptide E (residues 1-52) and peptide D (53-98), yielded the following NH2-terminal sequence for the H-2Kk molecule: [sequence in text]. Comparison of this sequence with those of other H-2 class I molecules revealed that: (1) Lys-19, Val-55, Glu-56, Asn-63 and Ile-73 are unique to the H-2Kk molecule; and (2) H-2Kk shares 79-83% homology in this region with other mouse class I molecules. Partial NH2-terminal amino acid sequences are also reported for the three disulfide-linked peptides. Several discrepancies from previously reported partial sequences of the H-2Kk molecule were detected.

Structural analyses define an additional H-2D region class I antigen that is expressed by a variant mouse strain.

The genetic complexity of the H-2D region includes haplotype disparities in apparent gene and product number. To probe the genetic basis of this complexity, the products of two independently derived mouse strains (STU and B10.SAA48) that express Dw3 antigens were compared. Serologic, fluorometric and peptide map comparisons were made using monoclonal antibodies. Although both STU and B10.SAA48 mice were found to express indistinguishable Dw3 molecules, only B10.SAA48 mice were found to express an additional antigen designated Lw3. Several lines of evidence are presented that suggest the gene encoding Lw3 maps to the D region. Furthermore peptide map comparisons of Dw3 with Lw3 molecules implied that they are products of separate genes; but Dw3 and Lw3 molecules were found to be more homologous to each other than Dd and Ld molecules are to each other. Inter-haplotype comparisons of Dw3 and Lw3 molecules with other D region molecules showed no striking homologies to Dd, Ld or eight other molecules compared. However, both Dw3 and Lw3 molecules were found to be unexpectedly homologous to the Ddx and Dw25 molecules, thus defining another family of structurally related D region antigens. This so called Dw3-family was found to be quite distinct from the previously defined Ld-family of molecules, since no joint members were found. The results of these studies of Dw3 encoded antigens are discussed as evidence for intra-D region recombination or mutation.

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 $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.

Virion-associated trans-regulatory protein of human T-cell leukemia virus type I.

Western blot analysis of HTLV-I virus particles from HUT-102 cells revealed a 40-kD protein strongly reactive with Tax-specific rabbit antisera. This protein subsequently was isolated from density gradient purified virions by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), purified from comigrating Gag and human cellular proteins by reversed-phase high-performance liquid chromatography (HPLC) and identified as the tax-encoded gene product by amino acid composition analysis. Among extracellular virions from five HTLV-I producing cell lines, only those from HUT-102 and C10MJ cells contained a detectable Tax protein, although all cells expressed Tax mRNA and protein intracellularly. To investigate the diagnostic implications of virion-associated Tax protein, sera from HTLV-I-infected individuals were compared on HUT-102 and MT-2 virus Western blots. The seroprevalence of antibodies to Tax, but not Gag or Env proteins, was substantially higher among adult T-cell leukemia and tropical spastic paraparesis patients using HUT-102 viral proteins. Thus, immunoassays utilizing HUT-102 virus are most sensitive for detection of Tax-reactive antibodies.

Chicken macrophages and thrombocytes share a common cell surface antigen defined by a monoclonal antibody.

A monoclonal antibody (mAb), designated K1, reacted with a cell surface antigen shared on chicken macrophages and thrombocytes. By immunofluorescence staining, the mAb K1 was reactive with 31.8% of peripheral blood lymphocytes (PBL) separated on Histopaque. In contrast, only 3.2% of PBL separated by slow-speed centrifugation were K1 positive. This antibody did not react with B- or T-lymphocytes, as demonstrated by the very small percentage of positive cells in thymus, bursa and spleen. Furthermore, no staining was observed with avian T-cell (MDCC-RP1 and SK3) or B-cell (LSCC-RP9) lines. Adherent cells derived from PBL separated on Histopaque and cultured for 48 h in plastic cell culture dishes were 81.5% positive with K1. These cells were also 82.6% positive with an antibody detecting the major histocompatibility complex (MHC) Class II antigen in chickens, indicating that they were monocyte-derived macrophages. Sheep red blood cell phagocytosis by these cells could be demonstrated, further supporting their macrophage lineage. In addition, K1 stained virtually 100% of HD11 cells, a chicken macrophage cell line, as well as 86.7% of peritoneal exudate cells. Eighty five percent of plastic adherent cells from PBL collected after 2 h of adherence reacted with the mAb K1, but only 8% of these cells were MHC Class II positive. These cells were morphologically identified as thrombocytes. Immunoprecipitation analysis demonstrated that the K1-reactive antigen consisted of a heterodimer with constituent polypeptide chains of 135 kDa and 61-68 kDa.

Molecular, cellular, and functional characterization of chicken cytokines homologous to mammalian IL-15 and IL-2.

DNA sequence analysis of a chicken interleukin (IL)-15 cDNA identified a 187 amino acid open reading frame encoding a protein with a predicted molecular weight of 21,964Da, two potential N-linked glycosylation sites, four highly conserved Cys residues, two out-of-frame AUG initiation codons in the 5' untranslated region, and an unusually long (66 amino acid) signal peptide such that the expected size of the mature protein is 14,462Da. Chicken IL-15 and IL-2 were compared with regard to their molecular, cellular, and functional characteristics. The predicted amino acid sequences of both chicken cytokines showed greater homologies with mammalian IL-15s compared with mammalian IL-2s. Northern hybridization and RT-PCR demonstrated chicken IL-15 gene transcripts in a wide variety of tissues and cell types while the chicken IL-2 gene was expressed only in concanavalin A (con A)-activated spleen cells. Both recombinant cytokines stimulated the growth of spleen T-cells and enhanced the activity of natural killer (NK) cells in vitro. Subcutaneous injection with an expression plasmid encoding IL-15 increased the percentage of CD3+ spleen T-lymphocytes whereas injection of an IL-2 cDNA augmented CD3+, CD4+, CD8+, T-cell receptor (TCR)1+, and TCR2+ T-cells. Collectively, these results indicate that chicken IL-15 and IL-2 are T-cell growth factors potentially capable of enhancing cell-mediated immunity in vivo.

Vaccines against the avian enteropathogens Eimeria, Cryptosporidium and Salmonella.

The worldwide poultry industry provides a substantial proportion of the nutritional requirement of the human population. To keep pace with the increasing demand for the high-quality, low-cost protein source that poultry provides, intensive rearing practices have been developed within the past few decades. For example, chickens are housed routinely in crowded environments under adverse conditions, and genetic strains have been selected for rapid growth, high protein-to-fat content and superior egg-laying characteristics. A major negative consequence of these practices has been an increase in the incidence of diseases. Enteric diseases in particular have emerged as a major problem threatening the future viability of the poultry industry. A variety of methods have been used to combat avian diseases in the commercial setting, including improved farm management practices, the use of antibiotic drugs, the selection of disease-resistant strains of chickens, and the manipulation of the chicken's immune system. In the latter category, the development of vaccines against the major avian diseases has become a priority in the poultry industry. This review will highlight recent progress in vaccine development against three major avian enteric pathogens: Eimeria, Cryptosporidium and Salmonella.

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.

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

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.

Quantitative differences in Ia antigen expression in the spleens of 15I5-B congenic and inbred chickens as defined by a new monoclonal antibody.

A monoclonal antibody (MAb), designated P2M11, that detects a monomorphic determinant of chicken class II antigens was produced from the fusion of P3X63 myeloma cells with spleen cells from BALB/c mice immunized with chicken splenic lymphocytes. Flow cytometric analyses of lymphocytes from the SC and FP strains of chickens showed 30 to 50% staining of bursa cells, 15 to 20% staining cells, and less than 5% staining of thymus cells. Addition of MAb P2M11 to splenic of T cell cultures stimulated with allogeneic cells or concanavalin A resulted in a significant inhibition of the T cell proliferation responses. Immunoprecipitation of 35S-methionine-labeled spleen cell extracts using MAb P2M11 identified molecules with apparent molecular weights of approximately 28,000, 30,000, and 32,000 by sodium dodecyl-polyacryl-amide gel electrophoresis. Taken together, these data indicate that the antigens detected by MAb P2M11 are similar in cell distribution and structure to chicken Ia antigens encoded by B-L genes. Using this MAb, a strain difference was demonstrated in the tissue distribution of Ia antigen positive lymphocytes in the spleens but not the thymuses of 15I5-B congenic and inbred strains of chickens. This difference may be due to the genes associated with B-complex genes.