A simple and efficient transfection protocol for Cryptosporidium parvum using Polyethylenimine (PEI) and Octaarginine.

The transfection of Cryptosporidium represents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection of Cryptosporidium parvum that takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of a Cryptosporidium species.

Apicomplexan co-infections impair with phagocytic activity in avian macrophages.

Mixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte-derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles ("Zymosan") by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella-infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to "Zymosan" was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

Expression of translationally controlled tumor protein (TCTP) gene of Dirofilaria immitis guided by transcriptomic screening.

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.

Identification of Dirofilaria immitis miRNA using illumina deep sequencing.

The heartworm Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariosis in dogs and cats, which also infects a wide range of wild mammals and humans. The complex life cycle of D. immitis with several developmental stages in its invertebrate mosquito vectors and its vertebrate hosts indicates the importance of miRNA in growth and development, and their ability to regulate infection of mammalian hosts. This study identified the miRNA profiles of D. immitis of zoonotic significance by deep sequencing. A total of 1063 conserved miRNA candidates, including 68 anti-sense miRNA (miRNA*) sequences, were predicted by computational methods and could be grouped into 808 miRNA families. A significant bias towards family members, family abundance and sequence nucleotides was observed. Thirteen novel miRNA candidates were predicted by alignment with the Brugia malayi genome. Eleven out of 13 predicted miRNA candidates were verified by using a PCR-based method. Target genes of the novel miRNA candidates were predicted by using the heartworm transcriptome dataset. To our knowledge, this is the first report of miRNA profiles in D. immitis, which will contribute to a better understanding of the complex biology of this zoonotic filarial nematode and the molecular regulation roles of miRNA involved. Our findings may also become a useful resource for small RNA studies in other filarial parasitic nematodes.

Characterisation and analysis of thioredoxin peroxidase as a potential antigen for the serodiagnosis of sarcoptic mange in rabbits by dot-ELISA.

BACKGROUND: Scabies caused by Sarcoptes scabiei is a widespread but a neglected tropical zoonosis. In this study, we characterised a S. scabiei thioredoxin peroxidase (SsTPx) and evaluated a recombinant SsTPx as a diagnostic antigen in rabbits. METHODS: The open reading frame of the gene encoding SsTPx-2 was amplified and the recombinant protein was expressed in Escherichia coli cells and purified. SsTPx was localized in mite tissue by immunolocalisation using the purified recombinant protein. Serodiagnosis assays were carried out in 203 New Zealand White rabbit serum samples by dot-ELISA. RESULT: The open reading frame (489 bp) of the gene encodes an 18.11 kDa protein, which showed highly homology to that of Psoroptes cuniculi (98.77% identity) and belongs to the 2-Cys family of peroxiredoxins. SsTPx was mainly distributed in muscle tissues of mites, integument of the epidermis and the anterior end of S. scabiei. Although SsTPx cross-reactivity with psoroptic mites was observed, the SsTPx dot-ELISA showed excellent diagnostic ability, with 95.3% sensitivity and 93.8% specificity in mange-infected and uninfected groups. CONCLUSIONS: This study showed that the purified SsTPx is a highly sensitive antigen for the diagnosis of mange infection by dot-ELISA. This technique is a rapid and convenient method that can be used worldwide for the clinical diagnosis of sarcoptic mange in rabbits, and is especially useful in developing regions.

Identification of neglected cestode Taenia multiceps microRNAs by illumina sequencing and bioinformatic analysis.

BACKGROUND: Worldwide, but especially in developing countries, coenurosis of sheep and other livestock is caused by Taenia multiceps larvae, and zoonotic infections occur in humans. Infections frequently lead to host death, resulting in huge socioeconomic losses. MicroRNAs (miRNAs) have important roles in the post-transcriptional regulation of a large number of animal genes by imperfectly binding target mRNAs. To date, there have been no reports of miRNAs in T. multiceps. RESULTS: In this study, we obtained 12.8 million high quality raw reads from adult T. multiceps small RNA library using Illumina sequencing technology. A total of 796 conserved miRNA families (containing 1,006 miRNAs) from 170,888 unique miRNAs were characterized using miRBase (Release 17.0). Here, we selected three conserved miRNA/miRNA* (antisense strand) duplexes at random and amplified their corresponding precursors using a PCR-based method. Furthermore, 20 candidate novel miRNA precursors were verified by genomic PCR. Among these, six corresponding T. multiceps miRNAs are considered specific for Taeniidae because no homologs were found in other species annotated in miRBase. In addition, 181,077 target sites within T. multiceps transcriptome were predicted for 20 candidate newly miRNAs. CONCLUSIONS: Our large-scale investigation of miRNAs in adult T. multiceps provides a substantial platform for improving our understanding of the molecular regulation of T. multiceps and other cestodes development.

Monocyte-Derived Chicken Macrophages Exposed to Eimeria tenella Sporozoites Display Reduced Susceptibility to Invasion by Toxoplasma gondii Tachyzoite.

Both Eimeria tenella and Toxoplasma gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. In this study, primary chicken monocyte-derived macrophages (MM) were infected with both pathogens to investigate mutual and host-parasite interactions. MM cultures were assigned to groups that were infected with E. tenella, T. gondii or both. In co-infected cultures, MM were first exposed to E. tenella sporozoites for 2 h. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 h post infection (hpi). Quantitative analysis for parasite replication was performed by real-time quantitative PCR (qPCR) at 2, 6, 12 and 24 hpi. Overall, the ability of T. gondii to penetrate the cell membrane of the potential host cell was reduced, although high motility was displayed. We found that T. gondii tachyzoites adhered for more than 4 h to macrophages during early co-infection. qPCR results confirmed that significantly less T. gondii entered in E. tenella-activated MM at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii, reducing active invasion and multiplication in chicken primary macrophages.

Mutual interactions of the apicomplexan parasites Toxoplasma gondii and Eimeria tenella with cultured poultry macrophages.

BACKGROUND: Toxoplasma gondii and Eimeria tenella are two common parasites in poultry. Mixed infections are likely to occur frequently in chickens due to the high prevalence of both pathogens. In this study, we investigate the co-occurrence of the two pathogens in the same immunocompetent host cell population towards potential parasite-parasite as well as altered patterns of parasite-host interactions. METHODS: Primary macrophages from chicken blood were co-infected in vitro with T. gondii tachyzoites (RH strain) and E. tenella sporozoites (Houghton strain) for 72 h. Morphological observations by light microscopy and assessments of parasite replication by quantitative real-time PCR (qPCR) were performed at 24, 48 and 72 h post-infection (hpi). Six host cell immune factors previously linked to T. gondii or E. tenella infection were selected for gene expression analysis in this study. RESULTS: Distinct morphological changes of macrophages were observed during mixed infection at 24 hpi and immunological activation of host cells was obvious. Macrophage mRNA expression for iNOS at 48 hpi and for TNF-α at 72 hpi were significantly elevated after mixed infection. Distinct upregulation of IL-10 was also present during co-infection compared to Eimeria mono-infection at 48 and 72 hpi. At 72 hpi, the total number of macrophages as well as the number of both parasites decreased markedly. As measured by qPCR, E. tenella population tended to increase during T. gondii co-infection, while T. gondii replication was not distinctly altered. CONCLUSIONS: Mutual interactions of T. gondii and E. tenella were observed in the selected co-infection model. The interactions are supposed to be indirect considering the observed changes in host cell metabolism. This study would thus help understanding the course of co-infection in chickens that may be relevant in terms of veterinary and zoonotic considerations.

Clinical efficacy of botanical extracts from Eupatorium adenophorum against the scab mite, Psoroptes cuniculi.

This study evaluated the in vivo clinical efficacy of Crofton weed (Eupatorium adenophorum) extracts against the scab mite, Psoroptes cuniculi. A 30-day experiment was performed using New Zealand rabbits that were naturally infested with P. cuniculi on a farm. Rabbits were randomly divided into five groups (6 animals per group); animals in groups A, B and C were treated in each ear topically with 2 ml of 1.0, 0.5 and 0.25 g/ml (w/v) E. adenophorum ethanol extract, respectively. Animals in groups D and E were treated with ivermectin (by injection; positive controls) and glycerol with water only (by embrocation; negative controls), respectively. Each rabbit was treated twice with separate treatments on days 0 and 7. Rabbits were observed daily and detailed examinations were performed on days 0, 7, 14 and 30, to inspect the presence or absence of mites and scabs/crusts. Clinical infection and the degree of recovery were evaluated, and the rate of reduction in mites and clinical efficacy rate (%) were calculated. The clinical effect of treatment with E. adenophorum extracts was similar to treatment with ivermectin. Seven days after the initial treatment, the mean clinical scores (presence of scabs/crusts) decreased from 3.32, 3.08 and 3.17 to 0.37, 0.47 and 0.48 in the left ears of animals in groups A, B and C, respectively, and from 3.53, 3.73 and 3.67 to 0.40, 0.45 and 0.48 in the right ears of animals in groups A, B and C, respectively, which were similar to the observations recorded in the positive control rabbits. However, the clinical score for negative control rabbits did not decrease significantly (P>0.05) during the experiment, and this changed from 3.32 to 2.75 in the left ears and from 3.50 to 3.25 in the right ears, and there were no significant differences in clinical efficacy between left and right ears. After two treatments (7 days space), the rabbits in groups A, B, C and D had recovered completely 30 days after the last treatment and no recurrences of infection were observed. These results indicate that E. adenophorum contains potent compounds for the effective control of animal acariasis.

Characterization and evaluation of a Sarcoptes scabiei allergen as a candidate vaccine.

BACKGROUND: Sarcoptic mange caused by the mite Sarcoptes scabiei is a worldwide disease affecting both humans and animals. Here we report the molecular characterization and evaluation of a recombinant S. scabiei tropomyosin (SsTm) protein in a vaccination trial in rabbits. METHODS: The full-length cDNA was cloned in a bacterial pET vector, and the recombinant protein was expressed in BL21 (DE3) cells and purified. Using specific rabbit antiserum, tropomyosin was localized immunohistochemically in mite tissue sections. Vaccination trials with the recombiant SsTm was carried out in New Zealand rabbits. RESULTS: The full-length open reading frame (ORF) of the 852 bp cloned gene from S. scabiei encodes a 32.9 kDa protein. The amino acid sequence showed 98.94%, 97.89% and 98.59% homology to Dermatophagoides farina and Dermatophagoides pteronyssinus group 10 allergens and Psoroptes ovis tropomyosin, respectively. Tropomyosin was localized immunohistochemically in mite tissue sections mainly in the mouthparts, legs and integument of the epidermis. The predicted cross-reactivity of SsTm indicated that it is an allergenic protein. While vaccination with the recombiant SsTm resulted in high levels of specific IgG (P < 0.01), a low IgE antibody response and no significant protection against S. scabiei challenge were observed. After challenge, specific IgG levels remained significantly higher than the control (P < 0.01), while changes of total IgE levels were not significant (P > 0.05). However, the lesion areas in the vaccination group decreased at the end of the experiment compared with controls. CONCLUSIONS: Although vaccination with recombinant SsTm did not efficiently control sarcoptic mange in rabbits, the immunogenic properties of tropomyosin suggest it may be developed as a vaccine with alternative adjuvants or delivery methods.

Detailed transcriptome description of the neglected cestode Taenia multiceps.

BACKGROUND: The larval stage of Taenia multiceps, a global cestode, encysts in the central nervous system (CNS) of sheep and other livestock. This frequently leads to their death and huge socioeconomic losses, especially in developing countries. This parasite can also cause zoonotic infections in humans, but has been largely neglected due to a lack of diagnostic techniques and studies. Recent developments in next-generation sequencing provide an opportunity to explore the transcriptome of T. multiceps. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a total of 31,282 unigenes (mean length 920 bp) using Illumina paired-end sequencing technology and a new Trinity de novo assembler without a referenced genome. Individual transcription molecules were determined by sequence-based annotations and/or domain-based annotations against public databases (Nr, UniprotKB/Swiss-Prot, COG, KEGG, UniProtKB/TrEMBL, InterPro and Pfam). We identified 26,110 (83.47%) unigenes and inferred 20,896 (66.8%) coding sequences (CDS). Further comparative transcripts analysis with other cestodes (Taenia pisiformis, Taenia solium, Echincoccus granulosus and Echincoccus multilocularis) and intestinal parasites (Trichinella spiralis, Ancylostoma caninum and Ascaris suum) showed that 5,100 common genes were shared among three Taenia tapeworms, 261 conserved genes were detected among five Taeniidae cestodes, and 109 common genes were found in four zoonotic intestinal parasites. Some of the common genes were genes required for parasite survival, involved in parasite-host interactions. In addition, we amplified two full-length CDS of unigenes from the common genes using RT-PCR. CONCLUSIONS/SIGNIFICANCE: This study provides an extensive transcriptome of the adult stage of T. multiceps, and demonstrates that comparative transcriptomic investigations deserve to be further studied. This transcriptome dataset forms a substantial public information platform to achieve a fundamental understanding of the biology of T. multiceps, and helps in the identification of drug targets and parasite-host interaction studies.

Annotation of the transcriptome from Taenia pisiformis and its comparative analysis with three Taeniidae species.

BACKGROUND: Taenia pisiformis is one of the most common intestinal tapeworms and can cause infections in canines. Adult T. pisiformis (canines as definitive hosts) and Cysticercus pisiformis (rabbits as intermediate hosts) cause significant health problems to the host and considerable socio-economic losses as a consequence. No complete genomic data regarding T. pisiformis are currently available in public databases. RNA-seq provides an effective approach to analyze the eukaryotic transcriptome to generate large functional gene datasets that can be used for further studies. METHODOLOGY/PRINCIPAL FINDINGS: In this study, 2.67 million sequencing clean reads and 72,957 unigenes were generated using the RNA-seq technique. Based on a sequence similarity search with known proteins, a total of 26,012 unigenes (no redundancy) were identified after quality control procedures via the alignment of four databases. Overall, 15,920 unigenes were mapped to 203 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Through analyzing the glycolysis/gluconeogenesis and axonal guidance pathways, we achieved an in-depth understanding of the biochemistry of T. pisiformis. Here, we selected four unigenes at random and obtained their full-length cDNA clones using RACE PCR. Functional distribution characteristics were gained through comparing four cestode species (72,957 unigenes of T. pisiformis, 30,700 ESTs of T. solium, 1,058 ESTs of Eg+Em [conserved ESTs between Echinococcus granulosus and Echinococcus multilocularis]), with the cluster of orthologous groups (COG) and gene ontology (GO) functional classification systems. Furthermore, the conserved common genes in these four cestode species were obtained and aligned by the KEGG database. CONCLUSION: This study provides an extensive transcriptome dataset obtained from the deep sequencing of T. pisiformis in a non-model whole genome. The identification of conserved genes may provide novel approaches for potential drug targets and vaccinations against cestode infections. Research can now accelerate into the functional genomics, immunity and gene expression profiles of cestode species.

Novel insights into the transcriptome of Dirofilaria immitis.

BACKGROUND: The heartworm Dirofilaria immitis is the causal agent of cardiopulmonary dirofilariosis in dogs and cats, and also infects a wide range of wild mammals as well as humans. One bottleneck for the design of fundamentally new intervention and management strategies against D. immitis may be the currently limited knowledge of fundamental molecular aspects of D. immitis. METHODOLOGY/PRINCIPAL FINDINGS: A next-generation sequencing platform combining computational approaches was employed to assess a global view of the heartworm transcriptome. A total of 20,810 unigenes (mean length  =  1,270 bp) were assembled from 22.3 million clean reads. From these, 15,698 coding sequences (CDS) were inferred, and about 85% of the unigenes had orthologs/homologs in public databases. Comparative transcriptomic study uncovered 4,157 filarial-specific genes as well as 3,795 genes potentially involved in filarial-Wolbachia symbiosis. In addition, the potential intestine transcriptome of D. immitis (1,101 genes) was mined for the first time, which might help to discover 'hidden antigens'. CONCLUSIONS/SIGNIFICANCE: This study provides novel insights into the transcriptome of D. immitis and sheds light on its molecular processes and survival mechanisms. Furthermore, it provides a platform to discover new vaccine candidates and potential targets for new drugs against dirofilariosis.

Acaricidal activity of extract from Eupatorium adenophorum against the Psoroptes cuniculi and Sarcoptes scabiei in vitro.

The possible acaricidal activity of Eupatorium adenophorum was analyzed using extracts created by water decocting, ethanol thermal circumfluence, and steam distillation. The toxic effect of each extract was tested against Psoroptes cuniculi and Sarcoptes scabiei in vitro. Ethanol thermal circumfluence extract had strong toxicity against mites, killing all S. scabiei at 0.5 and 1.0 g/ml (w/v) concentration, while 1g/ml extract was also found to kill all P. cuniculi within a 4-h period. Similarly, 0.25, 0.5 and 1.0 g/ml concentration of extract had strong toxicity against S. scabiei, with median lethal time (LT(50)) values at 0.866, 0.785 and 0.517 h, respectively. 0.5 g/ml and 1g/ml showed strong acaricidal action against P. cuniculi; the LT(50) values were 0.93 h and 1.29 h, respectively. The median lethal concentration (LC(50)) values were 0.22 g/ml for Scabies mite and 0.64 g/ml for P. cuniculi in 1h. The results indicated that E. adenophorum contains potent acaricidal ingredients; as a first step in the potential development of novel drugs, it may provide new acaricidal compounds for the effective control of animal acariasis.