Ostertagia ostertagi Mediates Early Host Immune Responses via Macrophage and Toll-Like Receptor Pathways.

Ostertagia ostertagi is an abomasal parasite with significant economic impact on the cattle industry. Early host immune responses are poorly understood. Here, we examined time course expression of Toll-like receptors (TLRs) in peripheral blood mononuclear cells (PBMC) during infection where PBMC macrophages (Mϕ) generated both pro- and anti-inflammatory responses when incubated with excretory/secretory products (ESP) from fourth-stage larvae (OoESP-L4) or adult worms (OoESP-Ad). First, changes in cell morphology clearly showed that both OoESP-L4 and OoESP-Ad activated PBMC-Mϕ in vitro, resulting in suppressed CD40 and increased CD80 expression. Expression of mRNAs for TLR1, -4, -5, and -7 peaked 7 days postinfection (dpi) (early L4), decreased by 19 dpi (postemergent L4 and adults) and then increased at 27 dpi (late adults). The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) (transcript and protein) increased in the presence of OoESP-Ad, and the anti-inflammatory cytokine interleukin 10 (IL-10) (protein) decreased in the presence of OoESP-L4 or OoESP-Ad; however, IL-10 mRNA was upregulated, and IL-6 (protein) was downregulated by OoESP-L4. When PBMC-Mϕ were treated with ligands for TLR4 or TLR5 in combination with OoESP-Ad, the transcripts for TNF-α, IL-1, IL-6, and IL-10 were significantly downregulated relative to treatment with TLR4 and TLR5 ligands only. However, the effects of TLR2 ligand and OoESP-Ad were additive, but only at the lower concentration. We propose that O. ostertagi L4 and adult worms utilize competing strategies via TLRs and Mϕ to confuse the immune system, which allows the worm to evade the host innate responses.

A simple molecular method to identify and quantify genera of gastrointestinal nematodes of cattle.

Classic approaches for antemortem identification of gastrointestinal nematodes (GIN) require coproculture of eggs and morphological examination. While adequate for diagnosis, many PCR techniques cannot easily quantify mixed infections without controls and/or standard curves. Herein, we developed a simple and rapid test for differentiating and quantifying mixed infections of GIN using PCR products separated by capillary electrophoresis. Among the cattle GIN, the ITS2 region is sufficiently distinct in length to delineate among the most common infecting genera, Ostertagia ostertagi = 373 bases (b), Haemonchus contortus (placei) = 366b, Cooperia punctata (oncophora) = 376b, Trichostrongylus axei = 372b, and Oesophagostomum radiatum = 357b. Conserved primers were synthesized that span the ITS2 where one primer was fluorescently labeled with 6-FAM. DNAs from infective L3 were PCR amplified then loaded onto an ABI 3130 sequencer adapted for size fragment analysis. Resulting peak amplitudes were both diagnostic and quantitative on a relative basis. As proof of principle, quantification was performed on PCR fragments from mixed species pairs of Ostertagia ostertagi, Cooperia punctata, and Haemonchus contortus and analyzed using Gene Marker V1.85 software. In all cases, linear responses were observed where R2 > 0.97 and line slopes ranged between 0.90 and 1.1. When tested on eggs from naturally infected animals, the assay showed superior results on two farms when compared to coproculture and morphological identification. Using wildlife-derived samples, results coincided well with deep amplicon sequencing. The assay is adaptable to large-scale studies, does not require comparative PCR controls, and should be compliant with GIN from small ruminant livestock.

Cholesterol of lipid rafts is a key determinant for entry and post-entry control of porcine rotavirus infection.

BACKGROUND: Lipid rafts are major structural components in plasma membranes that play critical roles in many biological processes including virus infection. However, few reports have described the relationship between lipid rafts and porcine rotavirus (PRV) infection. In this study, we investigated whether or not the locally high concentrations (3-5 fold) of cholesterol present in lipid rafts are required for PRV infection, and further examined which stages of the infection process are most affected. RESULTS: When cellular cholesterol was depleted by methyl-ß-cyclodextrin (MßCD), PRV infectivity significantly declined in a dose-dependent manner. This inhibition was partially reversed upon reintroduction of cholesterol into the system. This was corroborated by the co-localization of PRV with a recombinant, GPI-anchored green fluorescent protein, which functioned as a marker for membranous regions high in cholesterol and indicative of lipid rafts. Changes in virus titer and western blot analyses indicated that depletion of cellular cholesterol with MßCD had no apparent effect on PRV adsorption; however, depletion of cholesterol significantly restricted entry and post-entry of PRV into the cell. Both points of inhibition were restored to near normal levels by the addition of exogenous cholesterol. CONCLUSIONS: We conclude from these studies that membrane-based cholesterol and in particular that localized to lipid rafts, is an indispensable biomolecule for PRV infection, and that cholesterol-based control of the infection process takes place during entry and immediately post-entry into the cell.

Trichinella Nativa Outbreak With Rare Thrombotic Complications Associated With Meat From a Black Bear Hunted in Northern Ontario.

BACKGROUND: Although trichinellosis is known to cause thrombotic disease, serious thrombotic events are rare and have not been previously associated with Trichinella nativa infection. METHODS: Patient interviews and medical chart reviews were conducted on 10 men who became ill following consumption of a common source of black bear meat. Trichinella serology on patient sera as well as polymerase chain reaction (PCR) and larval identification of the meat samples was conducted. RESULTS: All 10 exposed individuals developed an acute illness clinically compatible with trichinellosis, characterized by fever, abdominal pain, and diarrhea, along with eosinophilia ranging from 0.9 × 109/L to 6.1 × 109/L. Within 2 weeks of the diarrheal illness, systemic symptoms developed in all exposed individuals characterized by fever, myalgia, periorbital edema, and fatigue. ST-elevation myocardial infarction and sinus venous tract thrombosis occurred as a complication of trichinellosis in 2 patients. Acute serology was nonreactive in all patients, though convalescent serology was reactive in 6 of 8 (75%) patients for whom sera was available. Multiplex PCR identified T. nativa from the bear meat, and was corroborated by microscopic larval identification. CONCLUSIONS: We report a 100% attack rate of T. nativa from bear meat among those who were exposed, and demonstrate that this species can cause serious thrombotic complications of trichinellosis in humans. Education of hunters and the public regarding the importance of proper preparation of wild game prior to ingestion is warranted.

Long-read sequencing improves assembly of Trichinella genomes 10-fold, revealing substantial synteny between lineages diverged over 7 million years.

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host-pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases.

Characterization of Ostertagia ostertagi annexin-like proteins at different developmental stages.

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host's gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P < 0.05) was higher in adult worms than in L3 or L4. The results indicate that the production of Oos-ANXL-2s is developmentally regulated and most abundant in the adult worm. This rather large family of proteins could be a potential vaccine target against O. ostertagi infection and warrants further investigation.

Phage display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential.

The spike (S) protein of porcine transmissible gastroenteritis virus (TGEV) is located within the viral envelope and is the only structural protein that possesses epitopes capable of inducing virus-neutralizing antibodies. Among the four N-terminal antigenic sites A, B, C, and D, site A and to a lesser extent site D (S-AD) induce key neutralizing antibodies. Recently, we expressed S-AD (rS-AD) in recombinant form. In the current study, we used the rS-AD as an immobilized target to identify peptides from a phage-display library with application for diagnosis. Among the 9 phages selected that specifically bound to rS-AD, the phage bearing the peptide TLNMHLFPFHTG bound with the highest affinity and was subsequently used to develop a phage-based ELISA for TGEV. When compared with conventional antibody-based ELISA, phage-mediated ELISA was more sensitive; however, it did not perform better than semi-quantitative RT-PCR, though phage-mediated ELISA was quicker and easier to set up.

Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity.

Porcine epidemic diarrhea virus (PEDV) is a pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in newborn piglets. Phage display is a technique with wide application, in particular, the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines. To identify antigen epitopes with specificity for PEDV, a monoclonal antibody (MAb-5E12) against the immunodominant region of the PEDV Spike protein (S1) was used as the target for biopanning a 12-mer phage display, random peptide library. After multiple rounds of biopanning and stringent washing, three phage-displayed peptides, designated L, W and H, were identified that recognize MAb-5E12. Sequence analysis showed that the one or more of the peptides exhibited partial sequence similarity to the native S1 sequence 'MQYVYTPTYYML' (designated peptide M) at position 201-212. In combination with software analysis for the prediction of B cell epitopes, aa 201-212 exhibited characteristics of a linear epitope on the PEDV S1 protein. In contrast to peptide M, a consensus motif 'PxxY' was identified on both peptides L and W, and on the S1 protein, but not on peptide H. Peptide M and the MAb-5E12-recognizing peptides L and W significantly inhibited the adsorption of PEDV on the cell surface as monitored through plaque-reduction assays. Furthermore, data from real-time PCR and indirect immunofluorescence assays were consistent with the ability of peptides M, L and W to block viral protein expression and thereby function as antiviral agents for PEDV.

Antiviral effect of diammonium glycyrrhizinate on cell infection by porcine parvovirus.

Porcine parvovirus (PPV) can cause reproductive failure in swine, resulting in economic losses to the industry. Antiviral effects of diammonium glycyrrhizinate (DG) have been reported on several animal viruses; however, to date it has yet to be tested on PPV. In this study, the antiviral activity of DG on swine testis (ST) cell infection by PPV was investigated using an empirically determined, non-toxic concentration of DG and three different experimental designs: (1) pre-treatment of virus prior to infection; (2) pre-treatment of cells prior to infection; and (3) direct treatment of virus-infected cells. The results showed that DG possesses potent inhibitory effects on PPV when the virus was treated before incubation with ST cells and that virus infectivity decreased in a dose-dependent manner. Results were confirmed by indirect immunofluorescence assays and real-time quantitative PCR. In addition, deoxycholate was used as a control to exclude the possibility that DG acted as a detergent to inhibit PPV infectivity. The study clearly indicates that DG has a direct anti-PPV effect in vitro.

Trichinella: Becoming a parasite.

Phylogenetic evidence indicates that free-living nematodes gave rise to parasitic nematodes where parasitism evolved independently at least 15 times. The high level of genetic and biological diversity among parasites dictates an equally high level of diversity in the transition to parasitism. We previously hypothesized that horizontal gene transfer (HGT) played an important role in the evolution of parasitism among early ancestors of Trichinella, mediated by an interplay of ecological and evolutionary pathways that contributed to persistence and diversification. We propose that host selection may have been associated with the metabolism of ammonia and engender a new paradigm whereby the reprogrammed nurse cell is capable of generating cyanate thereby enabling the importance of the Trichinella cyanase in the longevity of the cell. Parasites and parasitism have revealed considerable resilience against a backdrop of climate change and environmental perturbation. Here we provide a putative link between key periods in the evolution of Trichinella and major geological and climatological events dating back 500 million years. A useful lens for exploring such ideas, the Stockholm Paradigm, integrates Ecological Fitting (a foundation for host colonization and diversification), the Oscillation Hypothesis (recurring shifts between trends in generalization and specialization relative to host range), the Geographic Mosaic Theory of Coevolution (microevolutionary co-adaptive processes), and the Taxon Pulse Hypothesis (alternating events of biotic expansion i.e., exploitation in evolutionary and ecological time). Here we examine how one or more of these interactive theories, in a phylogenetic-historical context and in conjunction with HGT, may help explain the scope and depth of diversity among Trichinella genotypes.

The identification of small molecule inhibitors with anthelmintic activities that target conserved proteins among ruminant gastrointestinal nematodes.

Gastrointestinal nematode (GIN) infections are a major concern for the ruminant industry worldwide and result in significant production losses. Naturally occurring polyparasitism and increasing drug resistance that potentiate disease outcomes are observed among the most prevalent GINs of veterinary importance. Within the five major taxonomic clades, clade Va represents a group of GINs that predominantly affect the abomasum or small intestine of ruminants. However, the development of effective broad-spectrum anthelmintics against ruminant clade Va GINs has been challenged by a lack of comprehensive druggable genome resources. Here, we first assembled draft genomes for three clade Va species (Cooperia oncophora, Trichostrongylus colubriformis, and Ostertagia ostertagi) and compared them with closely related ruminant GINs. Genome-wide phylogenetic reconstruction showed a relationship among ruminant GINs structured by taxonomic classification. Orthogroup (OG) inference and functional enrichment analyses identified 220 clade Va-specific and Va-conserved OGs, enriched for functions related to cell cycle and cellular senescence. Further transcriptomic analysis identified 61 taxonomically and functionally conserved clade Va OGs that may function as drug targets for new broad-spectrum anthelmintics. Chemogenomic screening identified 11 compounds targeting homologs of these OGs, thus having potential anthelmintic activity. In in vitro phenotypic assays, three kinase inhibitors (digitoxigenin, K-252a, and staurosporine) exhibited broad-spectrum anthelmintic activities against clade Va GINs by obstructing the motility of exsheathed L3 (xL3) or molting of xL3 to L4. These results demonstrate valuable applications of the new ruminant GIN genomes in gaining better insights into their life cycles and offer a contemporary approach to discovering the next generation of anthelmintics.IMPORTANCEGastrointestinal nematode (GIN) infections in ruminants are caused by parasites that inhibit normal function in the digestive tract of cattle, sheep, and goats, thereby causing morbidity and mortality. Coinfection and increasing drug resistance to current therapeutic agents will continue to worsen disease outcomes and impose significant production losses on domestic livestock producers worldwide. In combination with ongoing therapeutic efforts, advancing the discovery of new drugs with novel modes of action is critical for better controlling GIN infections. The significance of this study is in assembling and characterizing new GIN genomes of Cooperia oncophora, Ostertagia ostertagi, and Trichostrongylus colubriformis for facilitating a multi-omics approach to identify novel, biologically conserved drug targets for five major GINs of veterinary importance. With this information, we were then able to demonstrate the potential of commercially available compounds as new anthelmintics.

*Inactivation of encysted muscle larvae of Trichinella spiralis in pigs using Mebendazole.

We evaluated the effect of 4 anthelmintic treatments on the viability of Trichinella spiralis encysted muscle larvae (ML) 55 days post infection (PI) in experimentally infected pigs. Muscle larvae were isolated from pig muscle by artificial digestion after oral treatment of pigs with Levamisole (8 mg/kg, daily for 5 days) and Mebendazole (50 mg/kg, daily for 5 days); Doramectin (0.3 mg/kg, single IM injection), and Moxidectin (0.5 mg/kg, single pour on). Isolated larvae from treated pigs were orally inoculated into mice to assess viability of ML from each treatment. Only Mebendazole treatment of pigs significantly reduced ML viability in mice. The effect of timing of the effective Mebendazole treatment on ML from a longer term infection was then examined in a second experiment. Analysis revealed that Mebendazole treatment of pigs with 250 mg/kg over 3 days (83 mg/kg/day) or 5 days (50 mg/kg/day) reduced numbers of ML recovered from pig tissues compared to untreated, infected controls, and rendered ML non-infective to mice; Mebendazole treatment of pigs with 250 mg/kg in a single dose was not effective in reducing ML numbers recovered from pigs or in impacting ML infectivity to mice. An examination of the lowest effective dose of Mebendazole on encysted ML was determined in a third experiment. Mebendazole of pigs with 5, 50, or 100 mg/kg over 3 days demonstrated that 5 or 50 mg/kg over 3 days insufficient to reduce infectivity in recovered ML, while 100 mg/kg (and 83 g from experiment 2) over 3 days significantly reduces infectivity of ML. This procedure provides a means to evaluate the efficacy of various anthelmintic treatments on the viability of Trichinella spiralis ML in pig tissues, and identified Mebendazole, at 83-100 mg/kg administered over a 3-5 day period as an anthelmintic which renders encysted Trichinella spiralis ML from pig tissues non-infective. As risk from Trichinella significantly impacts acceptance of pork from pasture-raised pigs, these data provide a method, especially for producers of these high-risk pigs, to eliminate the potential of Trichinella transmission from infected pork.

Transcriptome analyses reveal protein and domain families that delineate stage-related development in the economically important parasitic nematodes, Ostertagia ostertagi and Cooperia oncophora.

BACKGROUND: Cooperia oncophora and Ostertagia ostertagi are among the most important gastrointestinal nematodes of cattle worldwide. The economic losses caused by these parasites are on the order of hundreds of millions of dollars per year. Conventional treatment of these parasites is through anthelmintic drugs; however, as resistance to anthelmintics increases, overall effectiveness has begun decreasing. New methods of control and alternative drug targets are necessary. In-depth analysis of transcriptomic data can help provide these targets. RESULTS: The assembly of 8.7 million and 11 million sequences from C. oncophora and O. ostertagi, respectively, resulted in 29,900 and 34,792 transcripts. Among these, 69% and 73% of the predicted peptides encoded by C. oncophora and O. ostertagi had homologues in other nematodes. Approximately 21% and 24% were constitutively expressed in both species, respectively; however, the numbers of transcripts that were stage specific were much smaller (~1% of the transcripts expressed in a stage). Approximately 21% of the transcripts in C. oncophora and 22% in O. ostertagi were up-regulated in a particular stage. Functional molecular signatures were detected for 46% and 35% of the transcripts in C. oncophora and O. ostertagi, respectively. More in-depth examinations of the most prevalent domains led to knowledge of gene expression changes between the free-living (egg, L1, L2 and L3 sheathed) and parasitic (L3 exsheathed, L4, and adult) stages. Domains previously implicated in growth and development such as chromo domains and the MADF domain tended to dominate in the free-living stages. In contrast, domains potentially involved in feeding such as the zinc finger and CAP domains dominated in the parasitic stages. Pathway analyses showed significant associations between life-cycle stages and peptides involved in energy metabolism in O. ostertagi whereas metabolism of cofactors and vitamins were specifically up-regulated in the parasitic stages of C. oncophora. Substantial differences were observed also between Gene Ontology terms associated with free-living and parasitic stages. CONCLUSIONS: This study characterized transcriptomes from multiple life stages from both C. oncophora and O. ostertagi. These data represent an important resource for studying these parasites. The results of this study show distinct differences in the genes involved in the free-living and parasitic life cycle stages. The data produced will enable better annotation of the upcoming genome sequences and will allow future comparative analyses of the biology, evolution and adaptation to parasitism in nematodes.

Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus.

The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection.

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains.

BACKGROUND: Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. RESULTS: In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the last common ancestors (LCAs) of the three metazoan clades supports a correlation with organism adaptation. Furthermore, bursts of new protein families and domains in the LCAs of metazoans and vertebrates are consistent with whole genome duplications. CONCLUSION: Metazoan speciation and adaptation were explored by birth/death and duplication/deletion events among protein families and domains. Our results provide insights into protein evolution and its bearing on metazoan evolution.

Vaccination of mice with ORF5 plasmid DNA of PRRSV; enhanced effects by co-immunizing with porcine IL-15.

The open reading frame (ORF) 5 of porcine reproductive and respiratory syndrome virus (PRRSV) encodes a major envelope glycoprotein designated GP5. The GP5 protein is a candidate for vaccinating against PRRSV infection. In this study, recombinant plasmids bearing the PRRSV GP5 gene (pVAX-GP5) or the porcine interleukin 15 gene (pVAX-IL15) were generated. Mice were vaccinated with these gene constructs singularly or in combination, and subsequent humoral and cellular immune responses were evaluated. Proliferation assays showed that the number of T lymphocytes in the peripheral blood and spleens of treated mice were elevated by pVAX-GP5 and significantly enhanced by combination therapy involving pVAX-IL15. Flow cytometry data showed that the numbers of CD4+ and CD8+ T cells were also higher in treated mice. Both pVAX-GP5 treatment alone and in combination with pVAX-IL15 resulted in elevated antibody levels as demonstrated by indirect ELISA. The pVAX-IL15 gene construct served as a molecular adjuvant in conjunction with the pVAX-GP5 to enhance the immune responses where intermediate doses of pVAX-IL15 were most effective.

Horizontal gene transfer provides insights into the deep evolutionary history and biology of Trichinella.

Evolution involves temporal changes in the characteristics of a species that are subsequently propagated or rejected through natural selection. In the case of parasites, host switching also plays a prominent role in the evolutionary process. These changes are rooted in genetic variation and gene flow where genes may be deleted, mutated (sequence), duplicated, rearranged and/or translocated and then transmitted through vertical gene transfer. However, the introduction of new genes is not driven only by Mendelian inheritance and mutation but also by the introduction of DNA from outside a lineage in the form of horizontal gene transfer between donor and recipient organisms. Once introduced and integrated into the biology of the recipient, vertical inheritance then perpetuates the newly acquired genetic factor, where further functionality may involve co-option of what has become a pre-existing physiological capacity. Upon sequencing the Trichinella spiralis (Clade I) genome, a cyanate hydratase (cyanase) gene was identified that is common among bacteria, fungi, and plants, but rarely observed among other eukaryotes. The sequence of the Trichinella cyanase gene clusters with those derived from the Kingdom Plantae in contrast to the genes found in some Clade III and IV nematodes that cluster with cyanases of bacterial origin. Phylogenetic analyses suggest that the Trichinella cyanase was acquired during the Devonian period and independently from those of other nematodes. These data may help inform us of the deep evolutionary history and ecological connectivity of early ancestors within the lineage of contemporary Trichinella. Further, in many extant organisms, cyanate detoxification has been largely superseded by energy requirements for metabolism. Thus, deciphering the function of Trichinella cyanase may provide new avenues for treatment and control.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection.

The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell receptors and therefore specific cell types. It is also an important immune target for the host in neutralizing the virus. Four antigenic sites A, B, C, and D that reside near the N-terminal domain have been defined in the S protein. Of these, the region encoding antigenic sites A and to a lesser extent D, herein defined as S-AD, are most critical in eliciting host neutralizing antibodies. Herein, we enzymatically amplified, cloned, and expressed the S-AD fragment from TGEV in the prokaryotic expression vector, pET-30a. Maximum protein expression was achieved at 30°C over a 5-h period post-induction. Rabbit polyclonal antiserum was generated using recombinant S-AD (rS-AD) protein. In contrast to prior studies showing no activity with bacterially produced S protein, results indicated that polyclonal serum recognized TGEV-infected cells and reduced infection by 100%. Furthermore, the truncated rS-AD peptide was able to bind to the surface of cells from swine testes in a competitive manner and completely inhibit viral infection.

Repeated, drug-truncated infections with Ostertagia ostertagi elicit strong humoral and cell-mediated immune responses and confer partial protection in cattle.

Bovine ostertagiasis causes significant production losses to the cattle industry. Protective immunity induced by natural infection is slow to develop and anthelmintic resistance is rapidly developing. There is a need to advance alternatives for control of gastrointestinal nematode parasites. The present study investigated the effects of repeated, drug-truncated infections (rDTI) on development of protective immunity and attenuation of a challenge infection by O. ostertagi. Helminth-free calves were randomly assigned to either a rDTI or a control group (n = 5). The rDTI group received daily oral infections of 5000 Ostertagia L3 for 5 consecutive days, then were drug-treated on 14 and 15 days post infection (dpi), to attenuate O. ostertagi at the late fourth larval (L4) through young adult stages. DTI was repeated 3 weeks after the drug treatment. A total of 5 DTIs were administered to the DTI-treated animals. Non-DTI-treated, control animals received tap water as infection control. All animals were drug-treated at the same time. Animals were challenge-infected 4 weeks following the final round of rDTI. The results show that eggs per gram of feces (EPG) in the rDTI group were significantly reduced (P < 0.05) from 21 to 39 dpi, with an overall reduction in cumulative EPG. The control group exhibited reduced (P = 0.0564) average weight gains when compared to those of the rDTI group during weeks 4-5 post infection, a period coinciding with peak EPG output of control animals. Antigen-specific IgG, IgE and IgA responses were detected after the 2nd DTI, and stronger antibody recall responses were elicited by challenge infection. High levels of antigen-specific peripheral blood mononuclear cell (PBMC)/T cell proliferation to whole worm and excretory-secretory (ES) antigens were detected in rDTI-treated animals. These data indicate that partial protective immunity against ostertagiasis, involving cell-mediated and humoral responses, can be attained by rDTI which allowed for maximal antigen exposure from staggered parasitic developmental stages. The data suggest that rDTI can be used as a model to study host-parasite interactions and identify parasite antigens responsible for eliciting host protective immune responses.