Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.     

Interleukin-4 and IFN-gamma differentially stimulate macrophage chemoattractant protein-1 (MCP-1) and eotaxin production by intestinal epithelial cells.

When the intestine becomes infected by pathogenic organisms, intestinal epithelial cells (IEC) respond with the production of chemokines, which then attract and activate specific subsets of leukocytes. During chronic inflammation, the panel of IEC chemokines produced likely represents the net effect of a plethora of mediators present in the milieu, including cytokines from activated T lymphocytes. To explore the influence of T lymphocyte cytokines, we treated IEC-18 cells with interferon-y (IFN-gamma) and interleukin-4 (IL-4) and measured the effect on production of the CC chemokines, monocyte chemoattractant protein-1 (MCP-1) and eotaxin, and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Both IFN-gamma and IL-4 enhanced MCP-1 mRNA levels but with different kinetics. IFN-gamma stimulated a transient increase in MCP-1 mRNA levels, which peaked at 2 h, whereas IL-4-stimulated MCP-1 mRNA levels were markedly increased at 1 h and remained elevated at all time points studied. With each stimulus, the increase in MCP-1 mRNA levels was accompanied by a steady time-dependent increase in MCP-1 secretion. In addition, treatment with IFN-gamma or IL-4 enhanced IL-1beta-stimulated MCP-1 mRNA production and protein secretion. Eotaxin mRNA was detectable in unstimulated IEC-18 cells, and IL-4 but not IFN-gamma caused a rapid enhancement in levels, which remained elevated for 24 h after treatment. Finally, IL-1beta but not IFN-gamma or IL-4 enhanced MIP-2 mRNA levels. Knowledge gained from studying the outcome of T lymphocyte-derived stimuli will help understand the complex sequence of events during chronic intestinal inflammation.     

Differential chemokine response of murine macrophages stimulated with cytokines and infected with Listeria monocytogenes

During inflammatory processes the infected macrophage is a rich source of chemokines which induce infiltration of leukocytes to the site of infection. We investigated the regulation of chemokine production by murine macrophages in response to infection with the intracellular bacterial pathogen, Listeria monocytogenes. As a source of quiescent macrophages, murine bone marrow-derived macrophages (BMM) cultured under serum-free conditions were used. With RT-PCR, we detected induction of RNA message for the chemokines macrophage inflammatory protein (MIP)-2, KC, MIP-1alpha, MIP-1beta, IFN-gamma-inducible protein- 10 and RANTES in L. monocytogenes-infected macrophages. Accordingly, ELISA-detectable MIP-1alpha, MIP-2 and KC protein was induced by infection with L. monocytogenes. In contrast, L. monocytogenes infection of BMM alone failed to induce considerable expression of monocyte chemoattractant protein (MCP)-1 at the mRNA or protein level, but co-treatment with IFN-gamma was necessary. Release of infection- triggered MIP-2, MIP-1alpha and KC was negatively regulated by IFN- gamma. Similarly, IL-4 stimulated MCP-1 release by infected macrophages but reduced production of MIP-1alpha, MIP-2 and KC. IL-10 turned out to be a general deactivator in terms of macrophage chemokine production. IL-13 had no effect on MIP-1alpha, MIP-2 and KC production by infected BMM, but slightly reduced MCP-1 release. By using IFN-gamma and IL-4 gene deletion mutant mice, in vivo regulation of these chemokines by IL- 4 and IFN-gamma in listeriosis was studied. In summary, our results show that chemokines are produced by macrophages infected with L. monocytogenes, and that chemokine release is differentially regulated by the macrophage modulators IFN-gamma, IL-4, IL-10 and IL-13.      

Identification of seven Eimeria species in Swedish domestic fowl.

The aim of the study, conducted during the period 1992 to 1996, was to identify the Eimeria species present in Swedish chickens. All samples, including litter, faeces and guts from dead birds submitted for coccidial diagnosis, were obtained from farms where no live coccidiosis vaccines had ever been used. Identification of the different species was based on the criteria of oocyst morphology, location and characteristics of intestinal lesions, morphology of parasite endogenous stages, prepaient time and isoenzyme electrophoresis profiles of glucose phosphate isomerase. All seven Eimeria species of the domestic fowl were identified, namely E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella. Furthermore, Swedish monospecific isolates of E. maxima, E. necatrix and E. tenella were established.     

Eimeria: immunisation of young chickens kept in litter pens

Infection of chickens with an attenuated strain of Eimeria tenella gave good protection against challenge with the parent strain of this species. Chickens given an attenuated strain of E. acervulina var. mivati were protected against challenge with other pathogenic cultures of E. acervulina when kept in litter pens under conditions allowing reinfection. Two-week-old chickens, kept in litter pens, were given attenuated strains of E. tenella and E. acervulina and small numbers of oocysts (50-100) of E. brunetti, E. maxima and E. necatrix. Body weight gain was unaffected and resistance to challenge infection was demonstrated at 6 and 9 weeks after inoculation. In other experiments only a small proportion of 1 to 3-week-old chickens in each pen were given oocysts. Chickens placed in contact with these 'seeder' chickens were immunised against five species of Eimeria within a 2 to 4 week period. Similar experiments with 1-week-old chickens showed that these could also be immunised but the degree of parasitism during immunisation caused some weight gain depression. These results suggest that controlled immunisation against several species of Eimeria may be possible and further experiments are needed under conditions which more closely resemble those operating in the field.     

Toxoplasma gondii Infection of Neurons Induces Neuronal Cytokine and Chemokine Production, but Gamma Interferon- and Tumor Necrosis Factor-Stimulated Neurons Fail To Inhibit the Invasion and Growth of T. gondii

The intracellular parasite Toxoplasma gondii has the capacity to persist in the brain within neurons. In this study we demonstrated that T. gondii infected murine cerebellar neurons in vitro and replicated within these cells. Stimulation with gamma interferon (IFN-gamma) and/or tumor necrosis factor (TNF) did not enable neurons to inhibit parasite invasion and replication. Cultured neurons constitutively produced interleukin 1 (IL-1), IL-6, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta but not transforming growth factor beta1 (TGF-beta1), IL-10, and granulocyte-macrophage colony-stimulating factor. Neuronal expression of some cytokines (IL-6, TGF-beta1) and chemokines (MIP-1beta) was regulated by infection and/or by IFN-gamma and TNF.     

Common epitopes on Eimeria tenella sporozoites and cecal epithelium of chickens.

Eimeria tenella sporozoites invade the intestinal epithelium at a highly specific site. Certain molecules that were recognized by monoclonal antibody E.TEN 11M-2, which was prepared against E. tenella sporozoites, were detected on both cecal epithelium and sporozoites. We expect that these molecules are involved in attracting sporozoites towards the site of entry.     

特异性引物PCR鉴定鸡球虫种类与卵囊纯度的研究

为了建立鸡球虫种类的快速分子生物学鉴定方法,检测实验室保存虫株受污染状况,分别对单卵囊分离繁殖及实验室长期传代保存的6株巨型艾美耳球虫（EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01）、4株柔嫩艾美耳球虫（ETDS01、ETGD01、ETAD和ETAM）、1株堆形艾美耳球虫（EA1201）、1株变位艾美耳球虫（EMIS01）、1株毒害艾美耳球虫（ENGD01）收集卵囊,纯化、提取总DNA,根据巨型艾美耳球虫、柔嫩艾美耳球虫、堆形艾美耳球虫的RAPD和SCAR分子标记、ITS-1区序列分别设计Tn-F与Tn-R、Mx-F与Mx-R、Ac-F与Ac-R、ET-1与ET-2、EM-1与EM-2、EA-1与EA-2等6对特异性引物,对13个虫株分别进行PCR扩增,1%琼脂糖电泳分析片段大小.结果显示：用引物Tn-F与Tn-R、ET-1与ET-2对ETDS01、ETGD01、ETAD、ETAM、EMTO01扩增出特异性条带,其余4种8个虫株未见条带;用引物Mx-F与Mx-R、EM-1与EM-2对EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01扩增出特异性条带,其余4种7个虫株未见条带;用引物Ac-F与Ac-R、EA-1与EA-2对EA1201扩增出特异性条带,其余4种12个虫株未见条带.结果说明特异性引物PCR方法鉴定的5种球虫与原常规生物学方法鉴定的结果一致,检测出保存的巨型艾美耳球虫EMTO01虫株受柔嫩艾美耳球虫污染,其余虫株未发现交叉污染.研究证明利用特异性引物建立的PCR方法可用于鸡球虫种类与卵囊纯度的鉴定.     

Chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus

The goal of this study was to determine time courses of upregulation of several chemokines in nasal secretions after inoculation of human subjects with a low dose of live respiratory syncytial virus (RSV). Healthy, nonsmoking young adults were admitted to an inpatient clinical research unit. After baseline studies, subjects were nasally inoculated with approximately 10(3) plaque-forming units of RSV (strain A2), followed by daily nasal lavages. Nasal lavage fluid (NLF) was assayed for chemokines by specific ELISA. Of 10 subjects inoculated with RSV, 3 developed clinical symptoms of upper respiratory infection and also shed virus. Among infected subjects, there was a transient postinoculation increase in interleukin-8 (IL-8) in NLF to an average of 2.7-fold compared to baseline, followed by a prolonged increase (maximum mean 5.4-fold) during virus shedding. RANTES, MIP-1alpha, and MCP-1 all increased during virus shedding only (maximum mean increases of 5.3-fold, 13-fold, and 7.2-fold, respectively). Semiquantitative RT-PCR in brushed nasal epithelial cells on day 6 after inoculation suggested upregulation of RANTES, but not IL-8, mRNA during virus shedding. We conclude that chemokines IL-8, RANTES, MIP-1alpha, and MCP-1 are all increased in nasal secretions in human RSV infection at the time of virus shedding and symptomatic illness and that the epithelium lining the nasal turbinate contributes to the increase in RANTES.     

Selective induction of the monocyte-attracting chemokines MCP-1 and IP-10 in vesicular stomatitis virus-infected human monocytes.

It is characteristic of viral infections that monocytes/macrophages and lymphocytes infiltrate infected tissue, and neutrophils are absent. CC and non-ELR CXC chemokines predominantly attract mononuclear leukocytes, whereas the ELR motif-expressing CXC chemokines primarily act on neutrophils. To investigate the general role of chemokines in viral diseases, we determined their release and expression patterns after infection of human monocytes with vesicular stomatitis virus (VSV). Human monocytes were productively infected by VSV. Surprisingly, VSV did not induce the release of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6. In contrast, we found a strong induction of the CC chemokine monocyte chemotactic protein-1 (MCP-1) and the non-ELR CXC chemokine interferon-gamma (IFN-gamma) inducible protein-10 (IP-10) by VSV on the gene and protein level. The expression and release of the neutrophil chemoattractants IL-8 and growth-related oncogene-alpha (GRO-alpha) remained unaffected after VSV infection. Our results indicate that the typical monocyte and lymphocyte-dominated leukocyte infiltration of virus-infected tissue is based on a selective induction of mononuclear leukocyte-attracting chemokines.     

Expression of CINC-2beta is related to the state of differentiation of alveolar epithelial cells

Alveolar epithelial cells are among the first cells to encounter inhaled particles or organisms. These cells likely participate in the initiation and modulation of the inflammatory response by production of chemokines. However, there is little information on the extent or regulation of chemokine production by these cells. Rat type II cells were studied under differentiated and dedifferentiated conditions to determine their ability to express and secrete CXC chemokines. Both differentiated and dedifferentiated type II cells secreted MIP-2, MCP-1, and CINC-2 in response to a cytokine mixture of IL-1beta, TNF-alpha, and IFN-gamma or to IL-1beta alone. The cytokine mixture also induced iNOS expression and nitrite secretion. Both differentiated and dedifferentiated type II cells expressed CINC-1 (GRO), CINC-2alpha, CINC-3 (MIP-2), and MCP-1 mRNA, and their expression was increased by the cytokine mixture or by IL-1beta alone. However, CINC-2beta, a splice variant of CINC-2, was only expressed under differentiated conditions stimulated by KGF and was not increased by the cytokine mixture or by IL-1beta. In situ hybridization of normal lung and lung instilled with Ad-KGF demonstrated that CINC-2beta was expressed by alveolar and bronchiolar epithelial cells in vivo. We conclude that CINC-2beta is regulated differently from most other chemokines and that its expression is related to the state of alveolar type II cell differentiation.     

Relationship of coccidial lesion scores and weight gain in infections of Eimeria acervulina, E. maxima and E. tenella in broilers

A relationship was estimated between weight gains and coccidial lesion scores measured on individual male broiler chicks experimentally infected with different field isolates of Eimeria acervulina (seven tests), E. maxima (five tests) or E. tenella (eight tests). There was a small decrease in weight gain as lesion scores increased in E. acervulina infections, and a modest reduction in weight gain with increasing lesion scores in E. maxima and E. tenella infections. This relationship was observed in both nonmedicated birds and birds fed on diets containing 60 mg salinomycin/kg. The weight gains for birds fed 60 mg/kg with lesion scores of 2, 3 and 4 for E. acervulina. 1,2,3 and 4 for E. maxima and 2 and 3 for E. tenella were significantly greater than the weight gains of nonmedicated birds with the same lesion scores. The results demonstrated that lesion scoring does not fully reflect the degree of disease severity in induced infection. High lesion scores caused by the three species studied were associated with small changes in weight gain in medicated birds when compared with nonmedicated birds.     

Protein profiles of five avian Eimeria species.

SDS-PAGE fingerprint studies of oocyst antigens of five major Eimeria species including E. acervulina, E. maxima, E. necatrix, E. praecox and E. tenella demonstrated that their protein patterns are different, but there are some shared proteins between species and at least one protein band (45 kDa) was conserved among the five species. In Western blot studies, some species-specific as well as a few shared immunogenic bands were identified and chicken anti-E.maxima sera reacted with the conserved protein band in oocyst antigens of all these species.     

Identification of Eimeria species in Czechoslovakia

Coccidia isolated from 16 poultry farms in Czechoslovakia, were passaged once in chickens and then identified by microscopic examination of stages in different regions of infected gut, measurement of the oocysts and electrophoresis of lactate dehydrogenase and glucose phosphate isomerase. Two or more species of Eimeria were present concurrently on 14 farms and there was no substantial difference in the species recovered from poultry kept on litter or in cages. The most common species identified were, in decreasing order: E. acervulina (15/16 farms), E. tenella (11), E. mitis (8), E. maxima (6), E. praecox (5), E. brunetti (4) and E. necatrix (1).     

Intestinal intraepithelial lymphocytes sustain the epithelial barrier function against Eimeria vermiformis infection

Eimeria spp. are intracellular protozoa that infect intestinal epithelia of most vertebrates, causing coccidiosis. Intestinal intraepithelial lymphocytes (IEL) that reside at the basolateral site of epithelial cells (EC) have immunoregulatory and immunoprotective roles against Eimeria spp. infection. However, it remains unknown how IEL are involved in the regulation of epithelial barrier during Eimeria sp. infection. Here, we demonstrated two distinct roles of IEL against infection with Eimeria vermiformis, a murine pathogen: production of cytokines to induce protective immunity and expression of junctional molecules to preserve epithelial barrier. The number of IEL markedly increased when oocyst production reached a peak. During infection, IEL increased production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and decreased transforming growth factor beta (TGF-beta) production. Addition of IFN-gamma and TNF-alpha or supernatants obtained from cultured IEL from E. vermiformis-infected mice reduced transepithelial electrical resistance (TER) in a confluent CMT93 cell monolayer, a murine intestine-derived epithelial line, but antibodies against these cytokines suppressed the decline of TER. Moreover, TGF-beta attenuated the damage of epithelial monolayer and changes in TER caused by IFN-gamma and TNF-alpha. The expression of junctional molecules by EC was decreased when IEL produced a high level of IFN-gamma and TNF-alpha and a low level of TGF-beta in E. vermiformis-infected mice. Interestingly, IEL constantly expressed junctional molecules and a coculture of EC with IEL increased TER. These results suggest that IEL play important multifunctional roles not only in protection of the epithelium against E. vermiformis-induced change by cytokine production but also in direct interaction with the epithelial barrier when intra-EC junctions are down-regulated.