Rapamycin inhibits pathogen transmission in mosquitoes by promoting immune activation.

Repeated blood meals provide essential nutrients for mosquito egg development and routes for pathogen transmission. The target of rapamycin, the TOR pathway, is essential for vitellogenesis. However, its influence on pathogen transmission remains to be elucidated. Here, we show that rapamycin, an inhibitor of the TOR pathway, effectively suppresses Plasmodium berghei infection in Anopheles stephensi. An. stephensi injected with rapamycin or feeding on rapamycin-treated mice showed increased resistance to P. berghei infection. Exposing An. stephensi to a rapamycin-coated surface not only decreased the numbers of both oocysts and sporozoites but also impaired mosquito survival and fecundity. Transcriptome analysis revealed that the inhibitory effect of rapamycin on parasite infection was through the enhanced activation of immune responses, especially the NF-κB transcription factor REL2, a regulator of the immune pathway and complement system. Knockdown of REL2 in rapamycin-treated mosquitoes abrogated the induction of the complement-like proteins TEP1 and SPCLIP1 and abolished rapamycin-mediated refractoriness to Plasmodium infection. Together, these findings demonstrate a key role of the TOR pathway in regulating mosquito immune responses, thereby influencing vector competence.

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

Development of an immunocompetent mouse model susceptible to Cryptosporidium tyzzeri infection.

AIMS: Immunocompromised mice are extensively used in the screening of vaccines and drugs for Cryptosporidium, but this study model does not reflect the real status of infection in immunocompetent animals. This study aimed to provide an optimized animal model for future studies of Cryptosporidium vaccine. METHODS AND RESULTS: Three mouse strains (ICR, BALB/c and KM) with or without immunosuppression were compared after challenge with Cryptosporidium tyzzeri (C tyzzeri). The results indicated that ICR mice shed a greater number of faecal oocysts (20 346 ± 203 oocysts/g) compared with BALB/c (2077 ± 142 oocysts/g) and KM mice (3207 ± 431 oocysts/g) after experimental infection with C tyzzeri (P < .001). However, ICR mouse model is uniquely effective for C tyzzeri, not for other Cryptosporidium spp. such as C parvum. ICR mice were then used to determine the immunoreactions and immunoprotection of P23-DNA vaccine (pVAX1-P23) to C tyzzeri experimental infection. The results showed that a significant increase in anti-P23 antibody levels was induced by the pVAX1-P23 vaccine. Compared to pVAX1, TB and blank control mice, pVAX1-P23 immunized mice produced specific spleen cell proliferation as well as enhanced IL-5, IL-12p70 and IFN-γ production in sera. After challenge with 5 × 106 C tyzzeri oocysts, the oocyst shedding of the pVAX1-P23 immunized group was reduced by 69.94% comparing to the infection control. CONCLUSION: These results provide an optimized animal model for the study of prophylactic vaccines and this model might be applied to other candidates against Cryptosporidium, not only for pVAX1-P23.

Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 µg) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-γ and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 µg) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

CRISPR/Cas9 -mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection.

Plasmodium relies on numerous agonists during its journey through the mosquito vector, and these agonists represent potent targets for transmission-blocking by either inhibiting or interfering with them pre- or post-transcriptionally. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities for the study of agonist function and for developing malaria control strategies through gene deletion to achieve complete agonist inactivation. Here we have established a modified CRISPR/Cas9 gene editing procedure for the malaria vector Anopheles gambiae, and studied the effect of inactivating the fibrinogen-related protein 1 (FREP1) gene on the mosquito's susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants developed into adult mosquitoes that showed profound suppression of infection with both human and rodent malaria parasites at the oocyst and sporozoite stages. FREP1 inactivation, however, resulted in fitness costs including a significantly lower blood-feeding propensity, fecundity and egg hatching rate, a retarded pupation time, and reduced longevity after a blood meal.

Oral inoculation of ultraviolet-irradiated Eimeria species oocysts protects chickens against coccidiosis.

Prevention of coccidiosis is one of the best ways of controlling disease. Therefore, the present study was carried out to evaluate the protective effect of ultraviolet (UV)-irradiated sporulated oocysts of Eimeria species against coccidiosis in layer chickens. One hundred forty-four one-day-old layer chicks were randomly divided into 4 groups (n = 36), including non-immunized/non-challenged negative control group (NC group), non-immunized/challenged control group (NIC group), non-irradiated sporulated oocyst/challenged group (CA group), and UV-irradiated sporulated oocyst/challenged (UV group). At the age of 4 days, chickens in groups UV and CA were both orally inoculated with 1.0 × 104 UV-irradiated and non-irradiated sporulated oocysts of Eimeria species, respectively. Chickens in groups NIC and NC were served as positive and negative controls, respectively. Chickens in all groups were orally challenged with 7.5 × 104 sporulated oocysts of Eimeria species except the NC group at the age of 21 days. The results revealed that chicks receiving UV-irradiated sporulated oocysts had no signs of illness with minimal or no changes in the cecal integrity and a significantly lower oocyst shedding (OPG) than in the NIC group. Additionally, the cytokine gene expression profiles were evaluated. Expression levels of IL-2, IL-12, and IFN-γ were significantly higher in the spleen of chicks in the UV and CA groups than in the NC group post-challenge. As expected, treatment with irradiated oocysts resulted in a significant reduction in oocyst shedding and maintenance of cecal mucosal integrity. Furthermore, the body weight was higher in chickens inoculated with UV-irradiated oocysts than their non-irradiated counterparts. In conclusion, our results demonstrate that inoculation with UV-irradiated sporulated oocysts of Eimeria species can produce a substantial reduction in infection symptoms.

Selection and characterization of a precocious line of Eimeria media.

Coccidiosis, caused by the infection of Eimeria parasites, is one of the most common diseases in domestic rabbits. Live anticoccidial vaccine formulated with attenuated precocious lines of pathogenic eimerian parasites is expected to be valuable for the control of rabbit coccidiosis as a similar strategy to produce anticoccidial vaccines against chicken coccidiosis has being used for several decades. Eimeria media, moderate pathogenic, is widespread in China. Therefore, attenuated anticoccidial vaccines against rabbit coccidiosis should contain vaccine strain(s) of E. media. In this study, a precocious line of E. media (Empre) was selected by collecting and propagating the early excreted oocysts with 16 successive generations. The prepatent period of Empre reduced from 108 h of its parental strain (Emwt) to 70 h. The fecundity of Empre was about 1/10 to 1/3 lower than that of Emwt. Each sporocyst of Empre sporulated oocyst contained only one large refractile body instead of two smaller ones seen in the parental strain. When vaccinated with 1 × 103 or 1 × 104 precocious line oocysts, the rabbits were completely protected against homologous challenge with the parental strain 14 days post challenge by terms of body weight gain and oocyst output counting, indicating the efficacy of Empre. Meanwhile, all immunized rabbits showed no clinical sign post immunization, indicating the safety of Empre. For co-immunization, 1 × 103Empre oocysts and 5 × 102 oocysts of a precocious line of E. intestinalis (EIP8) were inoculated to each rabbit in a trial. No diarrhea or mortality was found after vaccination, and the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group, while the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group (P > 0.05), but significantly higher than that of UCC group (P < 0.01) after challenge, indicating it is safe and effective when using co-immunization. These results together show that Empre, as a precocious line, is a good candidate of precocious line of E. media for anticoccidial vaccine development.

Natural killer cell activity irreversibly decreases after Cryptosporidium gastroenteritis in neonatal mice.

Cryptosporidiosis causes persistent diarrhoea in infants, immunocompromised patients and elderly persons. Long-term consequences of the disease include increased risk of malignancy, cardiomyopathy and gastrointestinal inflammation. This study aimed to investigate prolonged effects of cryptosporidiosis on innate immunity and growth in neonatal C3HA mice. The disease was challenged by Cryptosporidium parvum oocyst inoculation into 7-day-old animals. The mice whose intestine smears contained 3-5 or 6 and more oocysts per microscopic field at the day 5 after infection were considered as mildly or severely infected, correspondingly. To determine natural killer cell (NK) activity, we applied 3 H-uridine cytotoxic assay to the animals at 5-68 days after infection using K562 cells as targets. At severe infection, there was a statistically significant 1.5-2.0 fold decline of body mass, spleen mass and spleen cellularity that persisted in animals of all ages. Accordingly, NK cytotoxicity showed even more drastic drop reaching 2.7-3.0 folds that was statistically significant in all animals. At mild infection, the discovered effects were less pronounced and reached significance only in some age groups. Thus, our study provides evidence that NK cells show long-term cytotoxic activity decrease following Cryptosporidium infection in neonatal mice, particularly in severe disease.

Immunoprotective responses against murine sarcocystosis by ß - Irradiated sporocysts.

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using ß-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 µSv ß-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. ß-irradiated sporocysts with 1.84 µSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

Isolation and biological and molecular characterization of Neospora caninum (NC-SP1) from a naturally infected adult asymptomatic cattle (Bos taurus) in the state of São Paulo, Brazil.

The biological and genetic diversity of Neospora caninum is very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate of N. caninum (NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog. Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences of N. caninum available in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developed N. caninum antibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.

Evaluation of the protective effect of pVAX-EtMIC3-recombined plasmid against E. tenella in chicken.

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial production worldwide. Micronemes of Eimeria play important roles in invading intestinal cell processes. In this study, the DNA vaccine expressing Eimeria tenella microneme protein 3 (EtMIC3) was constructed to evaluate its immune protective effect against E. tenella infection in chickens. The results demonstrated that chickens immunized with pVAX-EtMIC3 produced strong immune responses in the body, as shown by significant lymphocyte proliferation, cytokine production, and antibody responses. The average body weight gains of chickens in all the vaccinated groups were higher than those of non-vaccinated and challenged groups. In general, oocyst shedding was reduced, and bloody feces and gut lesion scores decreased. In addition, the survival rate of the immunized chickens increased compared to that of the unvaccinated and challenged control chickens. In summary, this study indicated that pVAX-EtMIC3 could induce protective immune effects against coccidiosis and that EtMIC3 is a potential vaccine candidate against coccidiosis.

Effect of threonine deficiency on intestinal integrity and immune response to feed withdrawal combined with coccidial vaccine challenge in broiler chicks.

For this study, threonine (Thr) deficiency was hypothesised to exacerbate the intestinal damage induced by feed withdrawal with coccidial infection because of its high obligatory requirement by the gut; two dietary Thr treatments (0·49 and 0·90 %) were applied to chicks from 0 to 21 d of age. At 13 d of age, feed was withdrawn for 24 h from one-half of birds of each dietary treatment with subsequent gavage of a 25× dose of coccidial vaccine. Overall, there were four treatments with eight replicate cages per treatment. Under combined challenge, birds fed the Thr-deficient diet had 38 % lower 13-21-d body weight gain (P≤0·05) compared with birds fed the Thr-control diet. At 21 d, the challenged group fed low Thr had higher number of oocysts (+40 %, P=0·03) and lower crypt depth (-31 %, P0·05). Overall, Thr deficiency worsened the detrimental effects of combined feed withdrawal and coccidial infection on growth performance and oocyst shedding by impairing intestinal morphology, barrier function, lymphocyte profiles and their cytokine expressions.

Salivary IgA against sporozoite-specific embryogenesis-related protein (TgERP) in the study of horizontally transmitted toxoplasmosis via T. gondii oocysts in endemic settings.

The aim of this study was to contribute to the better understanding of the relative epidemiological importance of different modes of infection with respect to horizontal transmission of Toxoplasma gondii in endemic settings. We investigated the prevalence of salivary IgA against a sporozoite-specific embryogenesis-related protein (TgERP) in a highly endemic area for toxoplasmosis in Brazil in order to pinpoint parasite transmission via oocysts. Prevalence calculated by salivary IgA specific to TgERP was compared to the prevalence calculated by serum IgG against both TgERP and tachyzoites (in conventional serological tests). Prevalence calculated by different serological and salivary parameters varied in the studied age groups. However, for the 15-21 years age group, values for T. gondii prevalence estimated by conventional serological tests and by anti-TgERP salivary IgA were similar; i.e. 68·7% and 66·6% or 66·7%, respectively, using two different cut-off parameters for salivary IgA anti-TgERP. Furthermore, salivary IgA anti-TgERP for this age group presented the highest specificity (93·33%), sensitivity (93·94%), and likelihood (14·09) compared to all the other age groups. These data demonstrate the importance of age for salivary IgA investigation against TgERP to estimate the mode of T. gondii transmission in endemic settings.

Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii.

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.

Innexin AGAP001476 is critical for mediating anti-Plasmodium responses in Anopheles mosquitoes.

The Toll and IMD pathways are known to be induced upon Plasmodium berghei and Plasmodium falciparum infection, respectively. It is unclear how Plasmodium or other pathogens in the blood meal and their invasion of the midgut epithelium would trigger the innate immune responses in immune cells, in particular hemocytes. Gap junctions, which can mediate both cell-to-cell and cell-to-extracellular communication, may participate in this signal transduction. This study examined whether innexins, gap junction proteins in insects, are involved in anti-Plasmodium responses in Anopheles gambiae. Inhibitor studies using carbenoxolone indicated that blocking innexons resulted in an increase in Plasmodium oocyst number and infection prevalence. This was accompanied by a decline in TEP1 levels in carbenoxolone-treated mosquitoes. Innexin AGAP001476 mRNA levels in midguts were induced during Plasmodium infection and a knockdown of AGAP001476, but not AGAP006241, caused an induction in oocyst number. Silencing AGAP001476 caused a concurrent increase in vitellogenin levels, a TEP1 inhibitor, in addition to a reduced level of TEP1-LRIM1-APL1C complex in hemolymph. Both vitellogenin and TEP1 are regulated by Cactus under the Toll pathway. Simultaneous knockdown of cactus and AGAP001476 failed to reverse the near refractoriness induced by the knockdown of cactus, suggesting that the AGAP001476-mediated anti-Plasmodium response is Cactus-dependent. These data demonstrate a critical role for innexin AGAP001476 in mediating innate immune responses against Plasmodium through Toll pathway in mosquitoes.

Comprehensive genetic dissection of the hemocyte immune response in the malaria mosquito Anopheles gambiae.

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca²âº channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.

Evaluation of an experimental irradiated oocyst vaccine to protect broiler chicks against avian coccidiosis.

The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, 1-day-old chicks were immunized with oocysts of Eimeria maxima, Eimeria acervulina, or Eimeria tenella exposed to gamma radiation ranging from 0-500 Gy. The litter oocyst counts at 7 days postimmunization, and the effect on weight gain following a challenge infection, decreased with an optimum dose between 150-200 Gy. Based on this finding, broiler chicks were immunized with a mixture of E. maxima, E. acervulina, and E tenella that had been exposed to 150 or 200 Gy. This resulted in more than a 100-fold reduction in litter oocyst counts and significant protection from a challenge infection, as measured by improved weight gain and feed conversion ratio (FCR). Immunization of birds with oocysts receiving 200 Gy was less effective in providing protection from a challenge infection. An additional formulation of vaccines containing two different oocyst doses of the three species that had been irradiated with 150 Gy were evaluated in their ability to attenuate oocyst output and convey protection to challenge. Results were similar with both high and low numbers of irradiated oocysts. Immunized chicks shed less oocysts at 7 days postimmunization and were protected from negative effects of challenge infection as measured by FCR, changes in weight gain, lesion scores, and measurement of body composition. However, the level of protection was somewhat less than that achieved by immunization with nonirradiated oocysts. The overall conclusion is that an irradiated oocyst vaccine developed in this study can effectively protect chicks that are raised on litter from challenge infection with multiple species of Eimeria, comparable to vaccines with virulent or precocious strains.

Biological characteristics of Chinese precocious strain of eimeria acervulina and its immune efficacy against different field strains.

In this study, the biologic characteristics of one experimental precocious strain of Eimeria acervulina and seven field isolates from different geographic locations in China were compared, and the immune efficacy of two precocious strains against coccidiosis in chickens was assessed to explore their potential use as coccidiosis vaccines. All the different strains were purified by single oocyst separation and their monospecificity was confirmed using E acervulina-specific PCR assays. The average sizes of E. acervulina oocysts were 18.28-20.19 X 14.09-14.79 microm and the shape indexes were from 1.28 to 1.40. The prepatent periods ranged from 93 to 115 hr, except for the Heyuan precocious strain (HYP; 75 hr). Chickens infected with Huadu field strain (GHD) produced the highest oocyst output whereas HYP induced the lowest level. When inoculated with 50,000 sporulated oocysts or more, the average weight gains of infected chickens were reduced, with apparent clinical symptoms. To assess the immunogenicity of precocious strains HYP and Baoding (BDP), birds were orally immunized and challenged with seven different field strains of E. acervulina. Body weight gain, fecal oocyst output, and gut lesion scores were compared to evaluate their vaccine potential. The results showed that the average body weight gains of chickens in all the vaccinated and challenged groups were higher than those of nonvaccinated and challenged groups. In general, oocyst shedding was reduced 34.39%-95.31% and gut lesion scores decreased 31.03%-86.21% compared with unvaccinated and challenged control chickens. In summary, this study indicated that the precocious strains of E. acervulina could induce a protective immune effect with various responses against coccidiosis caused by different field strains.