Immune synergistic mechanism of recombinant plasmid adjuvant containing chicken IL-4 and IL-2 fusion genes on chicken coccidia live vaccine.

The recombinant plasmid pCI-IL-4-IL-2-EGFP containing fusion genes of chicken IL-4 and IL-2 can be used as an adjuvant to enhance the anticoccidiosis effect of the chicken coccidia live vaccine. The chickens were divided into 3 groups: blank control group, vaccine + pCI-IL-4-IL-2-EGFP adjuvant coimmunization group, and vaccine-only group to investigate the immune synergy mechanism of recombinant plasmid adjuvant pCI-IL-4-IL-2-EGFP. The expressions of IL-2, IL-4, TNF-α, and IFN-γ in chicken sera and tissues were detected by ELISA and RT-qPCR, and the proliferation of T and B lymphocytes and antigen presenting cells (APC) in chicken immune organs and intestines were detected by acid alpha-naphthalase (ANAE) staining, methyl green pyronine (MGP) staining, and immunofluorescence (IF) staining, respectively. Results showed that the mRNA expression of IL-2, IL-4, IFN-γ and the number of activated T and B lymphocytes were significantly upregulated in the spleen and cecum tonsils of chickens in vaccine + pCI-IL-4-IL-2-EGFP group compared with the vaccine-only group on 7 d after vaccination (P < 0.05). Protein contents of IL-2, IL-4 and TNF-α in vaccine + pCI-IL-4-IL-2-EGFP group were significantly increased compared to vaccine-only group on 28 d of inoculation (P < 0.05). The number of T and B lymphocytes and APC in chickens of the vaccine+ pCI-IL-4-IL-2-EGFP group was significantly higher than that of the vaccine-only group in cecum tonsils, thymus and spleen after 14 and 28 d of inoculation (P < 0.05). All results revealed that pCI-IL-4-IL-2-EGFP adjuvant enhanced the immune response of chicken coccidia live vaccine by upregulating the expression of IL-2, IL-4, TNF-α, and IFN-γ and promoting the proliferation of T, B lymphocytes and APCs in chicken intestines and immune organ sites. Moreover, our study provides a theoretical basis for the clinical application of cytogenic plasmids as adjuvants.

A Coccidia-Specific Phosphate Transporter Is Essential for the Growth of Toxoplasma gondii Parasites.

Toxoplasma gondii is an obligate intracellular parasite that acquires all necessary nutrients from the hosts, but the exact nutrient acquisition mechanisms are poorly understood. Here, we identified three putative phosphate transporters in T. gondii. TgPiT and TgPT2 are mainly on the plasma membrane, whereas TgmPT is localized to the mitochondrion. TgPiT and TgmPT are widely present and conserved in apicomplexan parasites that include Plasmodium and Eimeria species. Nonetheless, they are dispensable for the growth and virulence of Toxoplasma. TgPT2, on the other hand, is restricted to coccidia parasites and is essential for Toxoplasma survival. TgPT2 depletion led to reduced motility and invasion, as well as growth arrest of the parasites both in vitro and in vivo. Both TgPiT and TgPT2 have phosphate transport activities and contribute to parasites' inorganic phosphate (Pi) absorption. Interestingly, the Pi importing activity of Toxoplasma parasites could be competitively inhibited by ATP and AMP. Furthermore, direct uptake of 32P-ATP was also observed, indicating the parasites' ability to scavenge host ATP. Nonetheless, ATP/AMP import is not mediated by TgPiT or TgPT2, suggesting additional mechanisms. Together, these results show the complex pathways of phosphate transport in Toxoplasma, and TgPT2 is a potential target for antitoxoplasmic intervention design due to its essential role in parasite growth. IMPORTANCE To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized. In this study, we identified two phosphate transporters, TgPiT and TgPT2, to localize to the plasma membrane of Toxoplasma. Although both TgPiT and TgPT2 possess phosphate transport activities, only the novel transporter TgPT2 was essential for parasite growth, both in vitro and in vivo. In addition, TgPT2 and its orthologs are only present in coccidia parasites. As such, TgPT2 represents a potential target for drug design against toxoplasmosis. In addition, our data indicated that Toxoplasma can take up ATP and AMP from the environment, providing new insights into the energy metabolism of Toxoplasma.

Genomic and Proteomic Evidence for the Presence of a Peroxisome in the Apicomplexan Parasite Toxoplasma gondii and Other Coccidia.

Apicomplexans are successful parasites responsible for severe human diseases including malaria, toxoplasmosis, and cryptosporidiosis. For many years, it has been discussed whether these parasites are in possession of peroxisomes, highly variable eukaryotic organelles usually involved in fatty acid degradation and cellular detoxification. Conflicting experimental data has been published. With the age of genomics, ever more high quality apicomplexan genomes have become available, that now allow a new assessment of the dispute. Here, we provide bioinformatic evidence for the presence of peroxisomes in Toxoplasma gondii and other coccidians. For these organisms, we have identified a complete set of peroxins, probably responsible for peroxisome biogenesis, division, and protein import. Moreover, via a global screening for peroxisomal targeting signals, we were able to show that a complete set of fatty acid ß-oxidation enzymes is equipped with either PTS1 or PTS2 sequences, most likely mediating transport of these factors to putative peroxisomes in all investigated Coccidia. Our results further imply a life cycle stage-specific presence of peroxisomes in T. gondii and suggest several independent losses of peroxisomes during the evolution of apicomplexan parasites.

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin.

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.

Nematicidal resorcylides from the aquatic fungus Caryospora callicarpa YMF1.01026.

This study investigated metabolites with activities against plant parasite nematodes from the fresh-water fungus Caryospora callicarpa YMF1.01026. We obtained three novel tetradecalactone metabolites, caryospomycins A-C, with such activities. The chemical structures of these were determined through NMR spectroscopic analysis and were found to belong to the 14-membered macrolides with a fused 1,2-dimethoxy-4-hydroxybenzene ring, a rare structure among the resorcylides. In the in vitro tests, all three compounds exhibited moderate killing activity against the nematode Bursaphelenchus xylophilus. To our knowledge, this is the first report of secondary metabolites in the aquatic fungal genus Caryospora.

Recent advances in the search for new anti-coccidial drugs.

Coccidia provide a rich hunting ground for drug-designers, as there are significant biochemical differences between the parasites and their hosts. Recent years have brought the discovery of the plastid and its possible metabolic machinery, characterisation of acidocalcisomes, reports on the apparent absence from some coccidia of a typical mitochondrion, and the discovery of the mannitol cycle and shikimate pathway in the parasites. Moreover, modern technologies such as genomics and proteomics are bringing new insights into the biochemistry of coccidia and highlighting possible drug targets in abundance. A major issue for would-be drug discoverers is to decide upon the targets to prioritise. This review provides an update on recent findings on how coccidia differ biochemically from vertebrates. It includes discoveries within coccidian parasites themselves but also uses findings in Plasmodium to provide an overview of biochemical features that may be characteristics of many apicomplexan parasites and so potential targets for broad-spectrum drugs.

Chemotherapeutic approaches to protozoa: Coccidiae--current level of knowledge and outlook.

Progress in the treatment and prophylaxis of cyst-forming coccidial infections (Neospora, Sarcocystis Toxoplasma) and Cryptosporidium infections has been limited (Table 1: Haberkorn 1996: Croft 1997: Wang 1997). However, new possibilities have been opened up in the treatment of Eimeria-induced coccidiosis in poultry and mammals. due to improvements in treatment and, or metaphylaxis. A new polyether antibiotic. semduramycin, has recently been added to the range of effective prophylactic preparations. The development of resistance to anticoccidial agents is now posing similar problems to those encountered with malaria, coccidiosis in poultry being particularly affected. Because no new active ingredient from a new family of chemical substances has been developed for more than 10 years, the following approaches are being adopted to get round this problem: the use of older preparations which have not been used for a long time, the introduction of combinations such as narasin nicarbazin or methyl benzoquate clopidol and the alternating use of anticoccidial agents in rotation and shuttle programmes. The goal of a real alternative, i.e. vaccination, has been achieved to a certain extent in the form of live vaccines for laying hens and broiler breeders and is being practiced in some countries.

Dietary betaine accumulates in the liver and intestinal tissue and stabilizes the intestinal epithelial structure in healthy and coccidia-infected broiler chicks.

The aim of this experiment was to study the patterns of betaine accumulation into intestinal tissue, liver and plasma of broiler chicks with or without coccidial infection. The chicks were raised on a corn-based, low-betaine diet with or without 1000 ppm betaine supplementation and with or without intestinal microparasite (Eimeria maxima) challenge to the age of 21 days. Plasma, liver, intestinal tissue and digesta of non-challenged (NC) birds and plasma and intestinal tissue of coccidiosis challenged (CC) birds were analysed for betaine content. NC birds were also analyzed for homocysteine in plasma and S-adenosylmethionine (S-AM) in liver. The jejunal epithelium was histologically examined for the presence of coccidia and the crypt-villus ratio was measured. Dietary betaine supplementation decreased the plasma homocysteine concentration but had no effect on liver S-AM of NC birds. The data suggest that chicks on a low-betaine diet accumulate betaine into the intestinal tissue. When the diet was supplemented with betaine, betaine accumulated heavily into liver and to a lesser degree into intestinal tissue. The concentration of betaine in jejunal and ileal digesta was low suggesting that dietary betaine was mainly absorbed from the proximal small intestine. The coccidial challenge decreased the concentration of betaine in the liver, but greatly increased that in the intestinal tissue. The crypt-villus ratio was decreased by the dietary betaine supplementation in healthy and challenged chicks, suggesting that dietary betaine both protects the jejunal villi against coccidial infection and also stabilizes the mucosal structure in healthy broiler chicks. These results support our earlier findings suggesting that betaine is likely to act as an important intestinal osmolyte in broiler chicks.

Labile neurotoxin in serum of calves with "nervous" coccidiosis.

Mouse inoculation was used to test for the presence of a toxin in the serum, cerebrospinal fluid, and intestinal contents collected from cases of bovine enteric coccidiosis, with and without neurological signs, and from control calves. Intravenous inoculation of mice with 10 mL/kg of serum from calves showing nervous signs caused effects significantly different from those caused by the inoculation of serum from calves not showing nervous signs and from control calves. The effect was particularly evident in female mice. At this dosage severe neurological signs such as loss of righting reflex, seizures and death occurred only with serum from calves with "nervous coccidiosis". The results suggest that serum from the calves with neurological signs contains a neurotoxin. This toxin appears to be highly labile. It was not present in the cerebrospinal fluid at levels comparable to those in the serum. The significance of this labile neurotoxin with respect to the pathogenesis of the neurological signs associated with bovine enteric coccidiosis is unknown.