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Giardia lamblia is a zoonotic protozoan that causes the diarrheal illness giardiasis, with the highest prevalence reported in the tropics and subtropics. Giardia is currently the most frequently identified pathogen in waterborne outbreaks in the United States. Nucleotide oligomerization domain (NOD) 1 and NOD2, intracellular NOD-like receptors, recognize pathogens to induce proinflammatory and antimicrobial responses. However, the roles of NOD1 and NOD2 signaling in Giardia infection have not yet been investigated. In the present study, the activation of NOD1 and NOD2 signaling pathways and the production of proinflammatory cytokines, reactive oxygen species (ROS) and nitric oxide in mouse macrophages stimulated with G. lamblia or parasite excretory-secretory products (ESPs) were examined. The results showed that G. lamblia and ESPs activated NOD2 and its downstream adaptor protein kinase, Receptor-interacting protein 2 (Rip2), in mouse macrophages. Blocking NOD2-Rip2 signaling significantly reduced the production of ROS and subsequently decreased the phosphorylation of nuclear factor-κB p65 and extracellular signal-regulated kinase, which in turn inhibited the production of four proinflammatory cytokines, namely, interleukin (IL)-1β, IL-6, IL-12p40 and tumor necrosis factor-α. In summary, our results indicate that the NOD2-Rip2 signal, which is activated by G. lamblia, contributes to the production of proinflammatory cytokines and ROS in mouse macrophages.

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2-/- mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3-/- mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2-/- and TLR3-/- mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2-/- and TLR3-/- mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2-/-, and TLR3-/- mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

The overall infection rate of Pentatrichomonas hominis in Siberian tigers in northeast China is 31.3%. All the P. hominis identified in Siberian tigers belonged to genotype CC1.

Autophagy has been shown to play an essential role in defending against intracellular bacteria, viruses, and parasites. Mounting evidence suggests that autophagy plays different roles in the infection process of different pathogens. Until now, there has been no conclusive evidence regarding whether host autophagy is involved in Neospora caninum infection. In the current study, we first monitored the activation of autophagy by N. caninum, which occurred mainly in the early stages of infection, and examined the role of host autophagy in N. caninum infection. Here, we presented evidence that N. caninum induced an increase in autophagic vesicles with double-membrane structures in macrophages at the early stage of infection. LC3-II expression peaked and decreased as infection continued. However, the expression of P62/SQSTM1 showed significant accumulation within 12 h of infection, indicating that autophagic flux was blocked. A tandem fluorescence protein mCherry-GFP-LC3 construct was used to corroborate the impaired autophagic flux. Subsequently, we found that N. caninum infection induced the activation of the TLR2–AKT–mTOR pathways. Further investigation revealed that TLR2–mTOR, accompanied by the blockade of autophagic flux, was responsible for impaired autophagy but was not associated with AKT. In vitro and in vivo, N. caninum replication was strongly blocked by the kinase inhibitor 3-methyladenine (3-MA, autophagy inhibitor). In contrast, rapamycin (Rapa, an autophagy inducer) was able to promote intracellular proliferation and reduce the survival rate of N. caninum-infected mice. On the other hand, the accumulation of autophagosomes facilitated the proliferation of N. caninum. Collectively, our findings suggest that activation of host autophagy facilitates N. caninum replication and may counteract the innate immune response of the host. In short, inhibition of the early stages of autophagy could potentially be a strategy for neosporosis control.

Background Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. Methods To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. Results Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 μg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. Conclusions Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage. Graphical abstract

Giardia duodenalis, a flagellated parasitic protozoan, the most common cause of parasite-induced diarrheal diseases worldwide. Codon usage bias (CUB) is an important evolutionary character in most species. However, G. duodenalis CUB remains unclear. Thus, this study analyzes codon usage patterns to assess the restriction factors and obtain useful information in shaping G. duodenalis CUB. The neutrality analysis result indicates that G. duodenalis has a wide GC3 distribution, which significantly correlates with GC12. ENC-plot result—suggesting that most genes were close to the expected curve with only a few strayed away points. This indicates that mutational pressure and natural selection played an important role in the development of CUB. The Parity Rule 2 plot (PR2) result demonstrates that the usage of GC and AT was out of proportion. Interestingly, we identified 26 optimal codons in the G. duodenalis genome, ending with G or C. In addition, GC content, gene expression, and protein size also influence G. duodenalis CUB formation. This study systematically analyzes G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB. These results will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution.

Background Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host’s innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored. Methods In this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs. Results The results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1β, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects. Conclusions The results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis–host interactions. Graphical abstract

Giardia duodenalis, also known as G. intestinalis or G. lamblia, is the major cause of giardiasis leading to diarrheal disease with 280 million people infections annually worldwide. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism participating in cells communications. The aim of this study is to explore the roles of G. duodenalis EVs (GEVs) in host-pathogen interactions using primary mouse peritoneal macrophages as a model. Multiple methods of electron microscopy, nanoparticle tracking analysis, proteomic assays, flow cytometry, immunofluorescence, qPCR, western blot, ELISA, inhibition assays, were used to characterize GEVs, and explore its effects on the host cell innate immunity as well as the underlying mechanism using primary mouse peritoneal macrophages. Results showed that GEVs displayed typical cup-shaped structure with 150 nm in diameter. GEVs could be captured by macrophages and triggered immune response by increasing the production of inflammatory cytokines Il1β, Il6, Il10, Il12, Il17, Ifng, Tnf, Il18, Ccl20 and Cxcl2. Furthermore, activation of TLR2 and NLRP3 inflammasome signaling pathways involved in this process. In addition, CA-074 methyl ester (an inhibitor of cathepsin B) or zVAD-fmk (an inhibitor of pan-caspase) pretreatment entirely diminished these effects triggered by GEVs exposure. Taken together, these findings demonstrated that GEVs could be internalized into mouse peritoneal macrophages and regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways., Author summary G. duodenalis, one of the most common cause of diarrheal diseases, is widely existed in the contaminated water and threatening the public health especially in developing countries. Along with the increasing resistance to anti-G. duodenalis drugs occurs, new targets against giardiasis are of urgently needed. The innate immune system is the first defense line of organism to resist multiple pathogens invasion through recognizing pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), termed Toll-like receptors (TLRs) on the surface of cell membrane and nucleotide oligomerization domain (Nod)-like receptors (NLRs) inside immune cells. Recently, extracellular vesicles have emerged as a ubiquitous mechanism participating in cells communications. In this study, EVs secreted by extracellular protozoan G. duodenalis were obtained and displayed typical cup-shaped structure with 150 nm in diameter. Moreover, GEVs could enter into primary mouse peritoneal macrophages and regulate host cell innate immunity by up-regulation of various inflammatory cytokines expression. Furthermore, TLR2 and NLRP3 inflammasome signaling pathways involved in this process. This study demonstrated that GEVs could be internalized into primary mouse peritoneal macrophages, regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways, and may provide new targets against giardiasis.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

Neospora caninum is an intracellular parasite that causes neosporosis in cattle. Bovine neosporosis is considered a major cause of bovine abortion worldwide. Rapid replication of N. caninum tachyzoites within host cells is responsible for the acute phase of N. caninum infection. Evidence shows that the host immune response plays an essential role in recognizing and regulating the replication of invading pathogens. Nucleotide-binding oligomerization domain receptors (NLRs) are a class of cytoplasmic sensors that can sense pathogens and induce the formation of the inflammasome complex. Activation of the inflammasome promotes restriction of microbial replication. Our previous study revealed NLRP3 inflammasome activation in N. caninum-infected murine macrophages. However, the role of inflammasome activity in N. caninum-infected bovine cells is unknown. To address this question, a bovine peritoneal macrophage cell line was used to investigate the role of inflammasome activation in regulating intracellular N. caninum replication. The results showed that inflammasome mediated activation of caspase-1 occurs in N. caninum-infected bovine macrophages, and caspase-1-dependent cell death was considered to be induced in N. caninum-infected bovine macrophages because N. caninum induced cell death decreased following pretreatment with zVAD-fmk and VX765. Meanwhile, the inhibition of caspase-1 in N. caninum-infected bovine macrophages led to the presence of more parasites in the parasitophorous vacuole. In contrast, inflammasome activation induced by ATP treatment in N. caninum-infected bovine macrophages contributed to the clearance of N. caninum. In addition, pyroptotic cell supernatant collected from ATP-stimulated bovine macrophages also impaired the ability of this parasite to infect new cells. In conclusion, this study is the first report on the role of the bovine inflammasome in restraining intracellular N. caninum replication and suggests that the bovine inflammasome may be a potential target for future development of drugs or vaccines against N. caninum infection in cattle.

Giardia lamblia is one of the most prevalent parasites residing in the duodenum of human and many other mammals throughout the world which is transmitted via ingested cysts through contaminated food or water. The severity of disease may depend on multiple parasite and host factors. Commonly, children and immunologically compromised persons like AIDS patient exhibit severe diarrhea, malabsorption and weight loss, however, there are also some infected people who are asymptomatic or only exhibit mild clinical symptoms and can shed the Giardia cysts in the environment. Although many studies have indicated that the innate immune system is important for Giardia defense, however, whether the innate immune responses such extracellular traps (ETs) could be induced by G. lamblia is still unclear. In recent years, macrophage extracellular traps (METs) have been described as an effective defense mechanism against invading microorganisms. In the present study, the formation of METs triggered by G. lamblia trophozoites was investigated. The formation of METs induced by G. lamblia trophozoites of mouse macrophage was observed with Scanning Electron Microscopy (SEM). The main components DNA, H3 histone and MPO were confirmed by Sytox orange staining, DNase1 digestion, immunofluorescence staining and fluorescence confocal microscopy. Inhibitor assays suggested that G. lamblia trophozoites triggered METs formation through ERK1/2 and p38 MAPK signal pathways and was Store-operated Ca2+ entry (SOCE) dependent. In addition, the process of METs formation triggered by G. lamblia trophozoites was also time and dose-dependent. Furthermore, the production of Reactive Oxygen Species (ROS) in macrophages stimulated with G. lamblia trophozoites significantly increased whereas no significant changes were observed about LDH activity.

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC50 ) of each tested drug to inhibit the germination of fungal spores. EC50 ranged between 0·7 and 47·2 μg ml-1 for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5-4057·8 μg ml-1 ). EC50 of tested fungicides was 0·6-2·3 μg ml-1 for carbendazim, 55·9-247·4 μg ml-1 for metalaxyl, 24·4-45·2 μg ml-1 for difenoconazole, and 555·9-1438·3 μg ml-1 for pentachloronitrobenzene (PCNB). EC50 of two antifungal drugs was 0·03-3·4 μg ml-1 for amphotericin B and 0·3-10·9 μg ml-1 for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.

Background Neospora caninum is an obligate intracellular protozoan that causes neosporosis, N. caninum infection is a major cause of abortion in cattle worldwide. Currently, specific treatment for neosporosis is not available. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a cytoplasmic protein complex that plays an important role in host defense against N. caninum infection, but the underlying mechanisms are poorly understood. Methods The reactive oxygen species (ROS) inhibitor and the ROS inducer, wild-type (WT) and NLRP3-deficient peritoneal macrophages or mice were used to investigate the role of ROS in NLRP3 inflammasome activation and controlling parasite burdens. ROS production, cell death and cell viability, production of inflammasome-mediated IL-1β or IL-18, cleavage of caspase-1 and NLRP3 expression, as well as parasite burdens were detected. Results In vitro, N. caninum induced ROS generation in a dose-dependent manner in peritoneal macrophages. The pretreatment of ROS inhibitor N-acetyl-l-cysteine (NAC) significantly attenuated N. caninum-induced ROS production, LDH release, IL-1β secretion and NLRP3 expression, whereas N. caninum proliferation was notably increased. In contrary, the ROS inducer pyrogallol (PG) significantly enhanced ROS production and NLRP3 inflammasome activity and decreased the parasite burden in N. caninum-infected peritoneal macrophages. NADPH-dependent ROS-mediated NLRP3 inflammasome activation induced by N. caninum can also be confirmed by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). However, the NAC or DPI pre-treatment or PG treatment did not significantly alter N. caninum-induced inflammasome activities and parasite proliferation in Nlrp3−/− peritoneal macrophages. In vivo, IL-18 releases in serum and parasite burdens in peritoneal exudate cells were significantly increased in PG-treated WT mice after infection with N. caninum; however, IL-18 productions and parasite burdens were not changed in PG-treated Nlrp3−/− mice. Furthermore, PG treatment in WT mice infected with N. caninum significantly decreased the mortality, weight loss and parasite burdens in tissues and histopathological lesions. Conclusions Neospora caninum-induced NADPH-dependent ROS generation plays an important role in NLRP3 inflammasome activation and controlling parasites. The ROS inducer PG can control N. caninum infection mainly by promoting NLRP3 inflammasome activation. ROS-mediated NLRP3 inflammasome axis can be a potential therapeutic target for neosporosis.

Neospora caninum (N. caninum) is an intracellular parasite and is the causative agent of neosporosis, which leads to reproductive failure in cattle. Pyroptosis is a recently discovered form of programmed cell death executed by gasdermin D (GSDMD). This cell death mechanism is an important host defense against intracellular pathogens. However, pyroptosis induced by N. caninum is poorly understood. The aim of this study was to explore the roles of GSDMD-mediated pyroptosis during N. caninum infection in vivo. N. caninum-infected wild type mice and GSDMD-deficient mice were used to evaluate host resistance and its ability to affect immune response against this parasite. The results showed that GSDMD deficiency significantly reduced survival and impaired the host's abilities to clear parasite loads in tissues, monocytes/macrophages and neutrophils. Additionally, GSDMD was essential for circulating IL-18 and IFN-γ production induced by N. caninum infection, indicating that GSDMD can mediate the Th 1 immune response against N. caninum infection. Additional data revealed that treatment with exogenous recombinant IL-18 in N. caninum-infected Gsdmd-/- mice rescues the reduction of circulating IFN-γ production to help eliminate the parasite. Taken together, our data indicate that GSDMD-mediated pyroptosis plays a vital role in maintaining host resistance to N. caninum and is essential for clearing the parasite. This form of programmed cell death promotes the Th 1 immune response by controlling IL-18 release and is considered a host defense against N. caninum. This study expands our understanding of interactions between host immune response/defense and N. caninum infection.

The present study was performed to investigate the seroprevalence and risk factors for Dirofilaria immitis infection in cats from Liaoning province, northeastern China. From October 2014 to September 2016, sera of 651 cats, including 364 domestic cats and 287 feral cats (332 females and 319 males) were assessed. They were tested for the presence of D. immitis antigen using SNAP Heartworm RT test kit. In this population, the average prevalence was 4.5%. Age and rearing conditions (feral or domestic) were found to be associated with the prevalence of D. immitis. The prevalence was significantly higher in feral cats compared with domestic cats (8.4% vs 1.4%, P0.05), but older cats (≥3 years old) showed a statistically higher prevalence compared with younger cats (0.05), all these results suggest that outdoor exposure time may be one of the most important factors for D. immitis prevalence in cats. Results reveal that D. immitis are prevalence in domestic and feral cats in northeastern China, which indicates that appropriate preventive measures should be taken to decrease the incidence of feline heartworm disease in Liaoning province, northeastern China.

The Apicomplexan parasite Neospora caninum is an obligate intracellular parasitic protozoan. It can cause severe diseases in a number of animals throughout the world. Infection with N. caninum leads to abortions in pregnant animals and neuromuscular disorders of newborns which cause great economic losses to animal husbandry. However, the mechanism of cell invasion by N. caninum is still unclear. This paper aims to investigate the impact of SB203580, a p38 MAPK inhibitor, on host cell invasion by N. caninum. The results suggested the presence of putative p38 MAPK homologues in N. caninum, and incubation of N. caninum with SB203580 markedly reduced the tachyzoite motility and microneme exocytosis (NcMIC2, 3, and 6). Furthermore, treatment or pretreatment of MDBK cells with SB203580 effectively reduced cell invasion by N. caninum. Therefore, SB203580 affected both, parasites and host cells, resulting in inhibition of cell invasion by N. caninum.

Toxoplasma gondii is an important zoonotic protozoan worldwide which infects most of warm-blooded mammals and birds, including human, and cause toxoplasmosis. As an intracellular parasite, T. gondii must evade host immune surveillance, such as IL-12 and IFN-γ, in order to survive and multiply in macrophages and other host cells. By delaying IL-12 secretion of host macrophages within 24 h after infection, T. gondii ensures not only self-survival but also the establishment of chronic infection of host cells. MicroRNA plays an important role in regulating gene transcription and translation. The mechanisms of IL-12 production during T. gondii infection are still unknown. Thus, understanding how the parasites manipulate IL-12 production by host macrophage is critical for the effective prevention and therapy of T. gondii infection. In the present study, regulation of delayed macrophage IL-12 production during T. gondii infection was explored. We found that the production of IL-12 after T. gondii infection was inhibited during the first 24 h and then resumed. The expression pattern of miR-187 production was consistent with the production pattern of IL-12 during T. gondii infection. The downregulation of miR-187 promoted Akt and P65 phosphorylation and delayed IL-12 production at late stage (after 24 h) of T. gondii infection. Dual-luciferase reporter assay indicated that MiR-187 targeted the NFKBIZ gene. Our results suggested that the delayed IL-12 production in mouse macrophages during T. gondii infection was regulated by miR-187.

Leishmaniasis, caused by the intracellular protozoan parasite Leishmania, remains an important neglected tropical infectious disease. Infection may be lethal if untreated. Currently, the available drugs for the disease are limited by high toxicity and drug resistance. There is an urgent need to develop novel anti-leishmanial strategies. Antimicrobial peptides (AMPs) have been described as the first-line immune defense against pathogenic microbes and are being developed as emerging anti-parasitic therapies. In the present study, we showed the anti-leishmanial activity of the synthetic 4-amino acid peptide lysine, aspartic acid, glutamic acid, and leucine (KDEL), the endoplasmic reticulum retention sequence, against Leishmania tarentolae promastigote and amastigote. Different concentrations of KDEL peptides were incubated with promastigotes, MTT viability assay, and promastigote assay were carried out. Macrophages infected with GFP-transfected L. tarentolae promastigotes were incubated with KDEL peptides, and the anti-amastigote activity of the KDEL peptides was measured by fluorescence microscopy. The damage of L. tarentolae was observed by light microscopy and electron microscopy. The cell apoptosis was analyzed using the Annexin V-FITC/PI apoptosis detection kit and mitochondrial membrane potential assay kit and by flow cytometry. Results showed that L. tarentolae was susceptible to KDEL peptides in a dose-dependent manner, and KDEL peptides disrupted the surface membrane integrity and caused cell apoptosis. In our study, we found for the first time an AMP KDEL from Pseudomonas aeruginosa and proved its significant therapeutic potential as a novel anti-leishmanial drug.

Trypanosome is considered as one of important parasites in almost all mammalian species, which occurs in Chiroptera throughout the world. Although numerous trypanosome species have been identified in bats in Asia, Africa, South America and Europe, little is known about the genetic diversity and pathogenicity of trypanosomes in Chinese bat. Recently, some human Trypanosoma cruzi infection attributed to a bat-related T. cruzi (TcBat) from the Noctilio spp., Myotis spp. and Artibeus spp was found. Consequently, it is a necessity to know trypanosome species in bats from China. In order to determine the prevalence and genotypes in bat from southwestern China, wehere detected trypanosomes prevalence 227bat brain tissue samples, including 60 Rousettus leschenaultia, 58 Hipposideros Pomona, 69 Rhinolophus pusillus, 40 Myotis daubentonni in Yunnan Province of China using nested PCR based on 18S rRNA. 14 (6.2%) of them were trypanosmes positive including 13 insect-eating bats and 1 fruit bat. The prevalence of trypanosome in R.leschenaultia, H. Pomona, and R.pusillus was 1.67%(1/60), 6.90%(4/58) and 13.0%(9/69), respectively (P 0.01), suggesting R. pusillus was a main-vector host bat. The positive rate of T.sp, T. dionisii, T.brucei brucei and T.sp ZY-2 was 4.8% (11/227), 0.4%(1/227), 0.4%(1/227), and 0.4% (1/227), resepectively. These results showed that T.sp-Yunnan is the predominant genospecies. To our knowledge, this is the first report about Trypanosome species in bats in Yunnan Province, southwestern China.

The intracellular parasite Toxoplasma gondii is a major cause of abortion and neonatal loss in livestock, and can cause severe illness to human with weakened immune system. The heavy incidence and severe consequence indicate the development of vaccines against T. gondii is required. In this study, DNA vaccines encoding GRA1 and MIC3 antigens were developed. The parasite-specific immune responses and protection efficiency against toxoplasmosis by these DNA vaccines were evaluated in BALB/c mice. The results demonstrated that the IgG antibody production was significantly increased in multi-antigenic vaccine encoding GRA1 and MIC3 immunized group, as well as the IFN-γ level, when compared with single-gene vaccines and controls groups (p 0.05). Two weeks after the final vaccination, the mice were challenged with either 1 × 10(4) or 1 × 10(2) RH strain tachyzoites, and the mortality and parasite reduction were observed. The multi-antigenic vaccine encoding GRA1 and MIC3 lead to the longest survival time as well as the less parasite-loads in brain and liver of immunized mice (p 0.01). The present study indicates that the GRA1 and MIC3 showed the potential as target for vaccine investigation against toxoplasmosis. And the immune efficacy induced by multi-antigenic vaccine encoding GRA1 and MIC3 was better than that induced by single-antigenic vaccines alone.

Avian coccidiosis makes a great threat and economic loss to the poultry industry, and fully understanding the innate immune response of chicken against E. tenella infection will play a significant role in avian coccidiosis prevention and treatment. Extracellular traps have been reported as a novel defense mechanism of host against pathogens infection. However, the interaction between chicken heterophil extracellular traps and E. tenella has remained not well known. Thus, this study aims to investigate the effects of E. tenella on chicken heterophil extracellular traps (ETs), and try to clarify the regulatory mechanisms in this process. E. tenella-triggered chicken heterophil ETs structures were analyzed by using scanning electron microscopy (SEM) and scanning confocal microscope. Inhibitors and Pico Green® were used to quantify E. tenella - triggered chicken heterophil ETs release. The results showed that E. tenella sporozoites significantly induced chicken heterophil ETs-like structures release, and histone and elastin co-existed with DNA in these structures of chicken heterophil ETs. Furthermore, it was also demonstrated that NADPH, p38 or Rac1 signaling pathways participated in E. tenella sporozoites-induced chicken heterophil ETs release, but more key molecules or signaling pathways involved in this process still needed to be further investigated. Taken together, this study reports that E. tenella sporozoites could induce chicken heterophil ETs formation via NADPH, p38 and Rac1 signaling pathways, which further suggests the critical role of heterophil ETs in the process of chicken against E. tenella infection.

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii that mainly infects canids as the definitive host and cattle as the intermediate host, resulting in abortion in cattle and leading to financial losses worldwide. Commercial vaccines or drugs are not available for the prevention and treatment of bovine neosporosis. Knowledge about the hallmarks of the immune response to this infection could form the basis of important prevention strategies. The innate immune system first responds to invading parasite and subsequently initiates the appropriate adaptive immune response against this parasite. Upon infection, activation of host pattern-recognition receptors expressed by immune cells triggers the innate immune response. Toll-like receptors, NOD-like receptors, and C-type lectin receptors play key roles in recognizing protozoan parasite. Therefore, we aimed to explore the role of the NLRP3 inflammasome during the acute period of N. caninum infection. In vitro results showed that N. caninum infection of murine bone marrow-derived macrophages activated the NLRP3 inflammasome, accompanied by the release of IL-1β and IL-18, cleavage of caspase-1, and induction of cell death. K+ efflux induced by N. caninum infection participated in the activation of the inflammasome. Infection of mice deficient in NLRP3, ASC, and caspase-1/11 resulted in decreased production of IL-18 and reduced IFN-γ in serum. Elevated numbers of monocytes/macrophages and neutrophils were found at the initial infection site, but they failed to limit N. caninum replication. These findings suggest that the NLRP3 inflammasome is involved in the host response to N. caninum infection at the acute stage and plays an important role in limiting parasite growth, and it may enhance Th1 response by inducing production of IFN-γ. These findings may help devise protocols for controlling neosporosis.

Cryptosporidium parvum is an important zoonotic parasite that causes significant economic loss in the animal husbandry industry, especially the cattle industry. As there is no specific vaccine or drug against Cryptosporidium, a rapid and accurate method for the detection of C. parvum is of great significance. In this study, colloidal gold strips were developed based on Cryptosporidium parvum virus 1 (CSpV1) for the detection of C. parvum infection in cattle fecal samples. The colloidal gold solution was prepared by reducing trisodium citrate and the CSpV1 #5 monoclonal antibody was labeled with colloidal gold. A polyclonal antibody against the CSpV1 capsid protein and an anti-mouse IgG antibody were coated on the colloidal gold strips for use in the test and control lines, respectively. Our results showed that the detection sensitivity in fecal samples was up to a 1:64 dilution. There was no cross-reaction with Cryptosporidium andersoni or Giardia in the fecal samples. The different preservation conditions (room temperature, 4°C, and 37°C) and preservation time (7, 30, 60, and 90 days) were analyzed. The data showed that the strips could be preserved for 90 days at 4°C and for 60 days at room temperature or 37°C. The colloidal gold strips were used to detect the samples of 120 clinical fecal in Changchun, China. The results indicated that the rate of a positive test was 5% (6/120). This study provides a rapid and accurate method for detecting C. parvum infection in cattle and humans.

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis. Previous studies have shown that the perturbation of mitochondrial metabolism in T. gondii results in growth deficiency in host cells and lack of virulence in animals. Members of this Letm1 protein family are inner mitochondrial membrane proteins which play a role in potassium and hydrogen ion exchange. Letm1 has not been characterized in T. gondii. In this study, a potential TgLetm1 gene (TgGT1_288400) with Letm1-like protein domain coding sequence was identified in T. gondii. Indirect immunofluorescence assays suggested that TgLetm1 localized to the mitochondria in tachyzoites, as indicated by the colocalization with mitochondrial marker Mitotracker. TgLetm1 was found in the membrane fraction by western blot analysis. To investigate the role of TgLetm1 in T. gondii, we generated a tetracycline-inducible TgLetm1-knock-down mutant. The conditional deletion of TgLetm1 resulted in mitochondrial swelling. Functional studies showed that the conditional deletion of TgLetm1 resulted in growth inhibition, deficiency in invasion and replication, and lack of virulence in mice.

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.

Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4.

Intestinal infection of pigs with trichomonads, (Tritrichomonas suis, Tetratrichomonas buttreyi, and Pentatrichomonas hominis) has been reported in many countries, such as the Philippines, Belgium, and the Czech Republic. However, the prevalence of trichomonads infection of swine in China has not yet been investigated. In this study, we used small subunit ribosomal RNA genes to detect the prevalence of T. suis, T. buttreyi and P. hominis among 158 fecal specimens from healthy pigs. Infection rates were 12.03%(19/158), 14.57% (23/158), and 24.05% (38/158) for T. suis, T. buttreyi and P. hominis, respectively. Molecular evolutionary analysis showed minor allelic variation in T. buttreyi from China compared to T. buttreyi isolated from other hosts in different parts of the world.

Background Neospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes. Methods Neospora caninum-infected wild-type (WT) macrophages and Nlrp3 −/− macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed. Results Many NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage. Conclusions The NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2197-2) contains supplementary material, which is available to authorized users.

Pentatrichomonas hominis is an anaerobic flagellated protozoan that primarily parasitizes the gastrointestinal tract and is a conditional pathogen. It has an extensive host range and is well known as a potential causative agent of zoonotic disease. The objective of this study was to provide the first findings of the prevalence of P. hominis in six farmed wildlife species, sika deer (S.D.), Rex rabbits (R.R.), blue foxes (B.F.), silver foxes (S.F.), raccoon dogs (R.D.) and minks (M.), that are commercially important in Jilin Province, China. In this study, 450 faecal samples were tested for P. hominis infection by culturing and nested PCR assays. The average prevalence of P. hominis infections were as follows: S.D. 20% (26/130), R.R. 16.25% (13/80), B.F. 45% (27/60), S.F. 43.33% (26/60), R.D. 53.33% (32/60) and M. 48.33% (29/60). The prevalence in herbivores (18.57% for S.D. and R.R.) was significantly lower than that in non-herbivores (47.5%). PCR product sequencing indicated that infections were mainly caused by the P. hominis strain Changchun Canine 1, and we found a P. hominis strain with a mutated sequence, Changchun-RR, which had three mutations compared with the referenced homologous P. hominis sequences. Morphological observations of the Changchun-RR strain showed that it was similar to P. hominis. Our study suggests that P. hominis is widespread in six farmed wildlife species in Jilin Province and provides baseline information for the presence of this parasite in these animals.

Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Dog is known as a definitive host of N. caninum and involved in transmitting infections to intermediate hosts by shedding oocysts. To investigate the epidemiology of dog neosporosis in China, 212 dog feces specimens in Shenyang were screened by nested-PCR using Nc5 primers and confirmed by N. caninum ITS1 PCR. The positive rate of N. caninum DNA was 34.90% (74/212). There were no significant correlations in prevalence of Neospora infections between different ages and genders. N. caninum DNA positive samples were further examined by PCR using Hammondia heydorni-specific primers. 37 out of 74 N. caninum DNA positive samples were also H. heydorni DNA positive. Only Nc5 primers positive and H. heydorni primers negative samples were used for ITS1 gene sequence analysis. Sequencing results from the 37 N. caninum positive samples revealed that ITS1 gene has 96-100% similarity with N. caninum sequences deposited in Genbank. Also, the presence of a new genotype indicated genetic polymorphism of N. caninum in infected dog feces in Shenyang of China.

Neospora caninum, an apicomplexan parasite, is recognized as a major bovine abortifacient. Dense granule antigens (GRAs) play important roles in the formation and modification of parasitophorous vacuoles (PVs) in Toxoplasma gondii. However, a few studies investigating GRAs have been reported in N. caninum. The aim of the present study was to characterize the dense GRA6/GRA7 of N. caninum in PVs using MDBK cells as a host cell model. Neospora caninum was inoculated into MDBK cells, and changes were observed using a transmission electron microscope (TEM). Neospora caninum GRA6/GRA7 were identified and characterized using bioinformatics, cell fractionation, and immunofluorescence. The TEM results revealed that integrated PVs were present in MDBK cells after N. caninum infection. Bioinformatics analysis showed that NcGRA6/NcGRA7 shared 28.76% and 29.66% homology with T. gondii GRA6/GRA7 (TgGRA6/TgGRA7) but had similar signal peptides, transmembrane domains, and motifs. Cell fractionation and subcellular localization analyses both showed that NcGRA6 was distributed in the lumen and intravacuolar network in soluble and transmembrane forms. The transmembrane form of NcGRA7 was observed in the PV membrane. These data lay a foundation for further study on bovine neosporosis and NcGRA6/NcGRA7 function during PV formation.

Background Neospora caninum is an apicomplexan parasite closely related Toxoplasma gondii, which causes neurological disease and abortion in multiple animal species. Macrophage polarization plays an important role in host immune responses to parasites infection, such as Toxoplasma gondii, Leishmania, Trypanosoma cruzi. However, the dynamics of macrophage polarization, as well as the possible mechanism that regulate macrophage polarization, during N. caninum infection remains unclear. Methods The M1 and M2-phenotypic markers of peritoneal macrophages from mice infected with tachyzoites of Nc-1 were analyzed by flow cytometry (FCM) analysis. Then J774A.1 cells were respectively treated with GW9662 and RGZ, and stimulated by tachyzoites of Nc-1. M1 and M2-phenotypic markers were determined by FCM and ELISA. And the activations of PPAR-γ and NF-κB were determined by Western blotting. Results In this study, our data showed that macrophages were preferentially differentiated into the M1 type during the acute stage of N. caninum infection, while the level of M2 macrophages significantly increased during the chronic stage of infection. In vitro study, compared with the GW9662 group and RGZ group, N. caninum can promote M2-polarized phenotype through up-regulate the activity of PPAR-γ and inhibting NF-κB activation. Conclusion In conclusion, this study demonstrated that macrophages are plastic since M1 differentiated macrophages can express M2 markers with N. caninum infection through up-regulating the activity of PPAR-γ and inhibting NF-κB activation and may be providing new insights for the prevention and treatment of N. caninum infection.

Toxoplasma gondii is an important intracellular parasite that is distributed worldwide and can infect almost all warm-blooded animals. The Chinese I genotype Wh3 strain is the most common in China and has unique pathogenicity compared with other strains of T. gondii. Bicoid-interacting protein (Bin3) is predicted to be involved in the development and polarity of T. gondii, and the localization of this protein is necessary for studying the biological characteristics of the Chinese I genotypeWh3 strain of T. gondii. In this study, we established an in vitro the method of transforming the tachyzoites into bradyzoites to lay the foundations for further experimental studies. Parasites were induced by culturing in alkaline conditions, then the changes in parasites morphology were evaluated. SAG2C, BAG1 and SAG1 were used to identify parasites. The results show that the Chinese I genotype Wh3 strain exhibited pseudocysts and cysts in the alkaline conditions after being induced, and the bradyzoite stage expressed specific proteins at the same time. Bradyzoites, induced using an alkaline medium (pH = 8.2), had higher expression levels of the Bin3 protein than tachyzoites. The results of indirect immunofluorescence, using a Bin3 monoclonal antibody showed that the Bin3 protein is expressed in both free-state and pseudocysts tachyzoites, and in the cysts of the Chinese I genotype Wh3 strain. The Bin3 protein is located in the cytoplasm of free-state tachyzoites, secreted between the parasite and the pseudocyst membrane in pseudocysts, and distributed inside the cyst wall of cysts. These findings provide a basis for further study on the biological characteristics of the Chinese I genotype Wh3 strain.

To assess the potential zoonotic transmission of giardiasis from dogs in China, a total of 205 fecal specimens from dogs were screened for Giardia duodenalis using PCR and sequence analysis of the triosephosphate isomerase gene. The prevalence of G. duodenalis in dogs was 13.2% (27/205). The potentially zoonotic assemblage A and the dog-specific assemblage C was identified in 25 (12.2%) and two (1.0%) dogs, respectively. All assemblage A isolates belonged to sub-assemblage AI, genotype AI-1. Likewise, one subtype was found in assemblage C. The high occurrence of potentially zoonotic G. duodenalis subtype AI-1 in dogs that are in close contact with humans is of public health concern.

Coccidiosis is one of the most important protozoan diseases and inflicts severe economic losses on the poultry industry. The aim of this study was to evaluate the capacity of Bacillus Calmette-Guerin (BCG) to deliver apical membrane antigen1 (AMA1) of Eimeria maxima to stimulate specific cellular and humoral immune responses in chickens. Day-old birds were immunized twice with rBCG/pMV261-AMA1, rBCG/pMV361-AMA1, or BCG via oral, intranasal, and subcutaneous routes and then orally challenged with homologous E. maxima sporulated oocysts. Gain of body weight, fecal oocyst output, lesion scores, serum antibody responses, numbers of splenocyte CD4(+) and CD8(+) T cells, and gut cytokine transcript levels were assessed as measures of protective immunity. Challenge experiments demonstrated that rBCG vaccination via intranasal or subcutaneous routes could increase weight gain, decrease intestinal lesions, and reduce fecal oocyst shedding, and the subcutaneous and intranasal routes were superior to the oral route based on the immune effects. Furthermore, intranasal rBCG immunization could also lead to a significant increase in serum antibody, the percentage of CD4+ and CD8+ T lymphocyte cells, and the levels of IL-1β, IFN-γ, IL-15, and IL-10 mRNAs compared with the control group. These results suggested that intranasal rBCG immunization could induce a strong humoral and cellular response directed against homologous E. maxima infection. This study provides data for the use of rBCG to develop a prophylactic vaccine against coccidiosis.

Background Docosahexaenoic acid (DHA) is a major dietary n-3 polyunsaturated fatty acid (n-3 PUFA) in fish oil, and has been reported to possess a number of biological properties, such as anti-inflammatory, antitumor and immune-regulatory properties. However, whether DHA exert anti-inflammatory effect on lipopolysaccharide (LPS)-induced mastitis remains unclear. In this study, we investigate the effect and underlying mechanisms of the effects of DHA on LPS-stimulated primary bovine mammary epithelial cells (bMEC). Methods The experiment was divided into six groups as followed: control group, GW9662 + LPS + DHA (100 μM) group, LPS and LPS + DHA (25, 50 and 100 μM) groups. bMEC were treated with DHA for 3 h before LPS (200 μg/ml) stimulation, and incubated with the PPARγ inhibitor GW9662 for 12 h before DHA treatment. The mRNA levels of TNF-α, IL-6 and IL-1β were measured by quantitative real-time PCR (qRT-PCR). Western blot was employed for measuring the transcriptional activity of NF-κB and PPARγ. Results Our results showed that DHA pretreatment significantly decreased the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in bMEC stimulated with LPS. Besides, DHA suppressed the phosphorylation of nuclear transcription factor-kappaB (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα) in NF-κB signal pathway, and activated proliferator activated receptor gamma (PPARγ). But, all those effects were obviously abolished by addition of GW9662, a specific inhibitor of PPARγ. Conclusion In conclusion, these results indicated that DHA may attenuate LPS-stimulated inflammatory response in bMEC by suppressing NF-κB activation through a mechanism partly dependent on PPARγ activation.

Eimeria stiedai (E. stiedai) is a coccidian that infects the liver of the domestic rabbit and may cause severe hepatic coccidiosis. Virus-like particles in E. stiedai were discovered by Revets et al. However, the complete genome sequence of the E. stiedai virus has yet to be determined. A novel virus was isolated from E. stiedai in the present study. The complete genome sequence of the E. stiedai virus was 6219 bp in length and contained two open reading frames (ORFs) with a tetranucleotide overlap (AUGA). ORF1 (2400 bp) encoded a putative coat protein of 799 amino acids (86.471 kDa) that exhibited a high level of amino acid sequence similarity to that of Eimeria tenella (E. tenella) RNA virus 1 (EtRV1; 43 % identity, NC_026140), whereas ORF2 (3303 bp) encoded a putative RNA-dependent RNA polymerase (RdRp) of 1100 amino acids (118.850 kDa) that exhibited a high level of amino acid sequence similarity to that of the E. tenella RNA virus 1 (EtRV1; 51 % identity, NC_026140). Phylogenetic analysis revealed that the E. stiedai virus was a new member of the family Totiviridae. The sequence data provided sufficient information for classification of eimeriaviruses.

Toxoplasmosis is one of the most world-wide spread zoonosis representing a very serious clinical and veterinary problem. There is still need for vaccines for toxoplasmosis. In the present study, we evaluated the protective efficacy of a recombinant actin depolymerizing factor (ADF) subunit vaccine against Toxoplasma gondii infection in BALB/c mice. The recombinant T. gondii ADF protein (rADF) was expressed in Escherichia coli and used as antigens for BALB/c mice immunization. The results indicated that specific antibody and the increased percentage of CD4(+) T lymphocyte were found in vaccinated BALB/c mice with rADF, when compared with adjuvant or PBS groups. After challenged with T. gondii (RH strain) tachyzoites, the survival time of the mice in rADF group was longer than those in the control group. The numbers of brain cysts of the mice in rADF group reduced significantly when compared with those in control groups (P0.05), and the rate of reduction could reach to around 30%. These results suggest that rADF can generate protective immunity against T. gondii infection in BALB/c mice.

The aim of the present study was to estimate the prevalence and risk factors of Dirofilaria immitis infection in dogs from Dandong, China. A total of 886 dogs were examined for D. immitis infection by microscopic examination and PCR, indicating that the prevalence was 16.6% (213/886) and 24.0% (147/886), respectively. The odds of infection were significantly higher in older dogs and dogs sheltered in outdoor, compared to the younger ones and ones sheltered in indoor. No significant difference of infection was observed in different genders, and between pure breed and cross-breed dogs in the same rearing conditions. These results indicated that the risk of exposure to D. immitis in dogs is high in Dandong, China, and prophylaxis against the parasite is advisable to decrease the incidence of canine dirofilariosis.

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4(+), CD8(+) T lymphocyte, and CD19(+) B lymphocyte were significantly increased (P0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.

Telomerase is essential for maintaining telomere length and chromosome stability in most eukaryotic organisms. The telomerase ribonucleoprotein complex consists of two essential components, the intrinsic telomerase RNA and the catalytic telomerase reverse transcriptase protein. Here we describe the cloning, sequencing and characterization of the telomerase reverse transcriptase catalytic subunit from the apicomplexan protozoon Eimeria tenella. The amino acid sequence predicted from it has all the signature motifs of the TERT family members. The E. tenella TERT cDNA contains an open reading frame encoding a protein with 1497 amino acids predicted size of 172 kDa and isoelectric point of 9.344. It contains the conserved reverse transcriptase motifs 1, 2, A, B, C, D and E as well as the TERT-specific T motif and the N-terminal conserved motifs GQ, CP and QFP. RT-PCR analysis indicated that E. tenella TERT mRNA expressed in sporozoites and merozoites phase while not in unsporulated oocysts. At the same time, the result of TRAP assay indicated that marked telomerase activity were detected in sporozoites and merozoites. In contrast, telomerase activity was not detected in unsporulated oocysts.

Cryptosporidium parvum is a protozoan parasite that infects a variety of mammals. The parasite has been shown to harbor a dsRNA virus (CPV) and in the present study, we have developed a CPV transient transfection system for this parasite by using green fluorescent protein (GFP) to replace the partial gene encoding region of the larger dsRNA (CPV-L) and the smaller dsRNA (CPV-S) virus. Two viral RNA-mediated transfection vectors: pCPVL-GFP and pCPVS-GFP were successfully constructed and both in vitro transcripts were electroporated into oocysts and sporozoites. Transient expression of GFP was detected in C. parvum oocysts and excysted sporozoites by fluorescence microscopy and by RT-PCR detection of GFP mRNA and antisense RNA in transfected C. parvum oocysts. Our study provides a new approach for studying gene expression and regulation in C. parvum and will hopefully lead to the construction of a stable CPV transfection system in the future.

Two recombinant Mycobacterium bovis BCG (rBCG) strains carrying the Eimeria tenella rhomboid gene (Rho) delivered by extrachromosomal vector pMV261 and integrative vector pMV361 were evaluated for their ability to protect chickens against E. tenella challenge. The chickens were immunized intranasal with BCG, rBCG pMV261-Rho, or rBCG pMV361-Rho twice at a 2-week interval. All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4(+) and CD8(+) cells compared to the control (P

Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry industry. Enhanced green fluorescent protein (EGFP) tagged recombinant Bacille Calmette-Guerin (rBCG), as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study. Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response. The colonization of rBCG in liver, spleen, lung, kidney and caecum was observed by laser confocal microscopy. Real-time quantitative RT-PCR showed a rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization. These results have significant implications for the development of rBCG vaccines against avian coccidiosis.

Giardia canis can be infected with a double-stranded RNA virus, that is giardiavirus (G. canis virus, GCV). In this study, green fluorescent protein (GFP) was stably expressed in G. canis mediated by GCV. The plasmid pNEO/GDH/MCS/GFP, containing the neomycin phosphotransferase (NEO) encoding region flanked by the 636 nt of 5'-terminus and the 2174 nt of 3'-terminus from GCV positive strand RNA, was constructed by inserting GFP gene into downstream from the NEO gene and glutamate dehydrogenase (GDH) 5'-terminus uncoding regions on a single plasmid, and its in vitro transcript was introduced into GCV-infected G. canis by electroporation. The transfectants expressed GFP persistently under G418 selection. This stable transfection system should provide a valuable tool for genetic study of G. canis.

A recombinant fowlpox virus (rFPV) expressing the Eimeria tenella rhomboid gene was constructed and its protective efficacy against homologous infection in chickens determined. Three-day-old-specific pathogen free (SPF) chickens were immunized s.c. with 10(2) plaque forming units (PFU), 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, and challenged with 5x10(4) homologous sporulated oocysts 14 days post-immunization (p.i.). The specific antibody response and lymphocyte proliferation were measured 1, 2, 3 and 4 weeks p.i. Oocyst output, body weight gains and lesion scores were measured to evaluate the protective effects of immunization. rFPV-rhomboid elicited a specific humoral immune response and stimulated proliferation of peripheral blood lymphocytes. The lesion scores in groups vaccinated with rFPV-rhomboid were significantly higher than in other groups. At the same time, rFPV-rhomboid improved body weight significantly compared with other groups. Immunization with rFPV-rhomboid reduced oocyst shedding significantly, resulting in a protection rate of 39.6%, 41.1% or 41.7% given a dose of 10(2) PFU, 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, respectively. These results indicated that rFPV can induce immune responses and offer partial protection of chickens against E. tenella challenge.

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.