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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Available evidence suggests that Trichinella spiralis first originated in Asia and subsequently spread to the rest of the world. Notably limited genetic diversity in European T. spiralis isolates indicates that the parasite went through a dramatic genetic bottleneck at some point in its history. Did this genetic bottleneck result from the transport of a limited number of T. spiralis infected pigs from Asian centers of domestication, or was the parasite resident in Europe far earlier than the domestication of pigs there? In order to explore this hypothesis, we generated complete mitochondrial genomes and ribosomal DNAs from seventeen European T. spiralis isolates, six North American isolates and seven Asian isolates using next generation sequencing. A total of 13,858 base pairs of mitochondrial DNA and 7431 nucleotides of the nuclear ribosomal DNA sequence from each isolate were aligned and subjected to phylogenetic analysis using T. nelsoni as an outgroup. We confirmed that North American and European isolates were tightly clustered within a single "western clade" and all Chinese T. spiralis isolates were placed within a well-supported sister clade. These results indicate that European T. spiralis did not directly descend from extant Chinese parasite populations. Furthermore, the amount of nucleotide divergence between the two clades suggests that they diverged before pigs were domesticated. Over evolutionary time periods, Chinese and European T. spiralis were likely maintained as separate populations. The data presented here indicates the genetic bottleneck observed in European T. spiralis did not result from a small number of founders introduced with Chinese pigs in the recent past, but derives from an earlier bottleneck in host populations associated with the end of the last glacial maximum., (Copyright © 2021. Published by Elsevier B.V.)

Bison ( Bison spp) are being reintroduced into semi-wild, spatially constrained herds across North America and Europe. Herd managers are concerned about gastrointestinal (GI) nematode parasites as they care for the health of their bison. We examine how demographics, grazing location, herd management, and anthelmintic treatments affect the fecal egg counts (FECs) of GI nematodes within a reintroduced Plains bison ( Bison bison bison ) herd in the Great Plains. Our results suggest that younger bison (<2 years of age) experience higher GI parasite eggs/oocysts per gram (epg/opg) and that some taxa are more prevalent throughout different periods of a bison's early years. Demographic findings suggest that calf and yearling (0-2 yrs age) bison have the highest FECs and that these decline until reaching a low in peak adulthood and thereafter (x > 6 yrs of age). FECs of both Trichuris spp. and particularly Nematodirus spp. were much more abundant, relatively, during the first year of a bison's life. This pattern was also true of Moniezia spp. and Eimeria spp., however, strongyle-type spp. FECs appeared to peak in relative abundance during the second year of life. Our data also indicate that FECs are influenced by differences in land-use histories of pastures previously grazed by cattle or by the proportion of frequent flooding in different pastures. Treatment results suggest that fenbendazole may more effective than moxidectin at lowering FECs of bison over the long-term, and lasting effects of at least one administered anthelmintic treatment. Multiplex PCR assays revealed that American bison share GI nematodes with cattle including: Ostertagia spp., Haemonchus placei, Cooperia onchophora , and Oesophagostomum spp, but did not detect the presence Trichostrongylus columbriformis . Our results may have wider conservation implications for reintroduction efforts of American bison, as well as the endangered European bison ( Bison bonasus )., (© 2021 The Authors.)

Gastrointestinal nematode (GIN) infections in cattle cause appetite suppression which leads to poor feed conversion, reduced weight gain and reduced milk production. Overuse and exclusive reliance on anthelmintic drugs has resulted in widespread resistance in many parasitic nematode species infecting livestock making control increasingly difficult. Wild ruminants are competent hosts of a number of nematode species that typically infect and are best adapted for cattle, sheep, and goats. Thus, the potential exists for wild ruminants to act as reservoirs in the translocation of domestic GIN, including those carrying anthelmintic resistance mutations as well as susceptible genotypes. The potential for parasite exchange is heightened by interfaces or ecotones between managed and wild rangelands, and by perturbations linked to climate warming that can increasingly alter the distributions of wild ungulates and their interactions with domestic and free-ranging ruminants. To investigate the extent to which wild ruminants harbour parasites capable of infecting domestic ruminants we first performed an epidemiological study of feces from wildlife hosts that spanned 16 states and included white-tailed deer (85 % of the samples), pronghorn, elk, mule deer, bighorn sheep, moose, cattle, and caribou across the United States. All samples were cultured to third stage larvae and nematode DNA was isolated and PCR amplified. Among the 548 wild ruminant samples received, 33 % (181 samples) were positive for nematode DNA, among which half (84 samples) contained DNA from GIN species commonly found in cattle. DNA from cattle GIN species was detected in 46 % of samples from the Northeast, 42 % from the Southeast, 10 % from the Midwest, 0 % from the Southwest and 11 % from the West. Deep amplicon sequencing of the ITS-2 rDNA indicated that Ostertagia and Trichostrongylus were present in 90 % and 69 % of the nematode DNA positive samples, respectively, whereas Haemonchus, Cooperia and Oesophagostomum were present in 26 %, 2 % and 10 % of the samples, respectively. These data clearly show that wild ruminants commonly harbour multiple parasite species whose primary hosts are domestic cattle, and suggest that further work is warranted to investigate their specific roles in the management of anthelmintic resistance., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Parasitic worms have a remarkable ability to modulate host immune responses through several mechanisms including excreted/secreted proteins (ESP), yet the exact nature of these proteins and their targets often remains elusive. Here, we performed mass spectrometry analyses of ESP (TsESP) from larval and adult stages of the pig whipworm Trichuris suis (Ts) and identified ~350 proteins. Transcriptomic analyses revealed large subsets of differentially expressed genes in the various life cycle stages of the parasite. Exposure of bone marrow-derived macrophages and dendritic cells to TsESP markedly diminished secretion of the pro-inflammatory cytokines TNFα and IL-12p70. Conversely, TsESP exposure strongly induced release of the anti-inflammatory cytokine IL-10, and also induced high levels of nitric oxide (NO) and upregulated arginase activity in macrophages. Interestingly, TsESP failed to directly induce CD4 + CD25 + FoxP3 + regulatory T cells (T reg cells), while OVA-pulsed TsESP-treated dendritic cells suppressed antigen-specific OT-II CD4 + T cell proliferation. Fractionation of TsESP identified a subset of proteins that promoted anti-inflammatory functions, an activity that was recapitulated using recombinant T. suis triosephosphate isomerase (TPI) and nucleoside diphosphate kinase (NDK). Our study helps illuminate the intricate balance that is characteristic of parasite-host interactions at the immunological interface, and further establishes the principle that specific parasite-derived proteins can modulate immune cell functions.

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-β-cyclodextrin (MβCD), 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 MβCD 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.

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.

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., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com)

Hybridization between two closely related but distinct genetic lineages may lead to homogenization of the two lineages with potentially novel phenotypes, or selective pressure to avoid hybridization if the two lineages are truly distinct. Trichinella nativa and Trichinella T6 are zoonotic nematode parasites which can be distinguished genetically despite occasional hybridization. Here, using an experimental murine model, we attempt to determine whether there are barriers to hybridization when sizeable numbers of each lineage are allowed to coinfect a host. Two mice were independently infected with equal numbers of T. nativa and T6. The offspring of these coinfections were genotyped at two microsatellite loci and one mitochondrial locus capable of distinguishing T. nativa from T6 genotypes. Among larvae in the F1 generation, offspring of every possible mating were encountered. Most larvae (63.6%) derived from T. nativa×T. nativa matings, while 21.1% of offspring were the product of T6×T6 matings, and only 15.3% were hybrid offspring of T. nativa×T6 crosses, differing markedly from null expectations. In this experimental model, T. nativa and Trichinella T6 were able to mate, but ratios of offspring indicated pre- or post-zygotic barriers to hybridization that may include assortative mating, genetic incompatibilities, and/or differences in the fitness of offspring. These barriers would limit gene flow between these two lineages in a natural setting, serving as a barrier to their homogenization and promoting their persistence as distinct and separate entities., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

During the United States Department of Agriculture (USDA) National Animal Health Monitoring System's (NAHMS) 2007-2008 beef study, 567 producers from 24 US States were offered the opportunity to collect fecal samples from weaned beef calves and have them evaluated for the presence of parasite eggs (Phase 1). Participating producers were provided with instructions and materials for sample collection. Up to 20 fresh fecal samples were collected from each of the 99 participating operations. Fresh fecal samples were submitted to one of 3 randomly assigned laboratories for evaluation. Upon arrival at the laboratories, all samples were processed for the enumeration of strongyle, Nematodirus, and Trichuris eggs using the modified Wisconsin technique. The presence or absence of coccidian oocysts and tapeworm eggs was also noted. In submissions where the strongyle eggs per gram exceeded 30, aliquots from 2 to 6 animals were pooled for DNA extraction. Extracted DNA was subjected to genus level polymerase chain reaction (PCR) identification for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. In this study, 85.6% of the samples had strongyle type, Nematodirus, and Trichuris eggs. Among the samples evaluated, 91% had Cooperia, 79% Ostertagia, 53% Haemonchus, 38% Oesophagostomum, 18% Nematodirus, 7% Trichuris, and 3% Trichostrongylus. The prevalence of coccidia and tapeworm eggs was 59.9% and 13.7%, respectively.

During the United States Department of Agriculture (USDA) National Animal Health Monitoring System's (NAHMS) 2007-2008 beef study, producers from 24 states were offered the opportunity to evaluate their animals for internal parasites and for overall responses to treatment with anthelmintics. A lapse of 45 d was required between initial sampling and any previous treatments. Choice of anthelmintic (oral benzimidazoles, and both injectable and pour-on endectocides) was at the discretion of the producer so as not to alter the local control programs. Fresh fecal samples were collected from 20 animals, or from the entire group if less than 20, then randomly assigned to 1 of 3 participating laboratories for examination. Analyses consisted of double centrifugation flotation followed by enumeration of strongyle, Nematodirus, and Trichuris eggs (the presence of coccidian oocysts and tapeworm eggs was also noted). Where strongyle eggs per gram (epg) exceeded 30, aliquots from 2 to 6 animals were pooled for egg isolation and polymerase chain reaction (PCR) analysis for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. Results from 72 producers (19 States) indicated that fecal egg count reductions were < 90% in 1/3 of the operations. All operations exhibiting less than a 90% reduction had used pour-on macrocyclic lactones as the anthelmintic treatment. While some of these less than expected reductions could have been the result of improper drug application, PCR analyses of the parasite populations surviving treatment, coupled with follow-up studies at a limited number of sites, indicated that less than expected reductions were most likely due to anthelmintic resistance in Cooperia spp. and possibly Haemonchus spp.

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.

The distribution, abundance, and diversity of life on Earth have been greatly shaped by human activities. This includes the geographic expansion of parasites; however, measuring the extent to which humans have influenced the dissemination and population structure of parasites has been challenging. In-depth comparisons among parasite populations extending to landscape-level processes affecting disease emergence have remained elusive. New research methods have enhanced our capacity to discern human impact, where the tools of population genetics and molecular epidemiology have begun to shed light on our historical and ongoing influence. Only since the 1990s have parasitologists coupled morphological diagnosis, long considered the basis of surveillance and biodiversity studies, with state-of-the-art tools enabling variation to be examined among, and within, parasite populations. Prior to this time, populations were characterized only by phenotypic attributes such as virulence, infectivity, host range, and geographical location. The advent of genetic/molecular methodologies (multilocus allozyme electrophoresis, polymerase chain reaction-DNA [PCR-DNA] fragments analysis, DNA sequencing, DNA microsatellites, single nucleotide polymorphisms, etc.) have transformed our abilities to reveal variation among, and within, populations at local, regional, landscape, and global scales, and thereby enhanced our understanding of the biosphere. Numerous factors can affect population structure among parasites, e.g., evolutionary and ecological history, mode of reproduction and transmission, host dispersal, and life-cycle complexity. Although such influences can vary considerably among parasite taxa, anthropogenic factors are demonstrably perturbing parasite fauna. Minimal genetic structure among many geographically distinct (isolated) populations is a hallmark of human activity, hastened by geographic introductions, environmental perturbation, and global warming. Accelerating environmental change now plays a primary role in defining where hosts, parasites, and other pathogens occur. This review examines how anthropogenic factors serve as drivers of globalization and genetic homogenization of parasite populations and demonstrates the impact that human intervention has had on the global dissemination of parasites and the accompanying diseases.

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.

Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by porcine epidemic diarrhea virus (PEDV). However, when PEDV, TGEV and porcine pseudorabies virus were incubated with peptide H (HVTTTFAPPPPR), only infection of Vero cells by PEDV was inhibited. Immunofluoresence assays indicated that inhibition of PEDV infection by peptide H was independent of pAPN. Western blots demonstrated that peptide H interacted with PEDV spike protein and that pre-treatment of PEDV with peptide H led to a higher inhibition than synchronous incubation with cells. These results indicate direct interaction with the virus is necessary to inhibit infectivity. Temperature shift assays demonstrated that peptide H inhibited pre-attachment of the virus to the cells., (Copyright © 2014. Published by Elsevier Inc.)

Pseudorabies virus (PRV) is a double-stranded, DNA-based swine virus with a genome approximating 150 kb in size. PRV has many nonessential genes which can be replaced with genes encoding heterologous antigens but without deleterious effects on virus propagation. Recombinant PRVs expressing both native and foreign antigens are able to stimulate immune responses. In this paper, we review the current status of live attenuated recombinant PRVs and live PRV-based vector vaccines with potential for controlling viral infections in animals.

Contrary to our understanding of just a few decades ago, the genus Trichinella now consists of a complex assemblage of no less than nine different species and three additional genotypes whose taxonomic status remains in flux. New data and methodologies have allowed advancements in detection and differentiation at the population level which in turn have demonstrably advanced epidemiological, immunological and genetic investigations. In like manner, molecular and genetic studies have permitted us to hypothesise biohistorical events leading to the worldwide dissemination of this genus, and to begin crystalising the evolution of Trichinella on a macro scale. The identification of species in countries and continents otherwise considered Trichinella-free has raised questions regarding host adaptation and associations, and advanced important findings on the biogeographical histories of its members. Using past reviews as a backdrop, we have ventured to present an up-to-date assessment of the taxonomy, phylogenetic relationships and epidemiology of the genus Trichinella with additional insights on host species, survival strategies in nature and the shortcomings of our current understanding of the epidemiology of the genus. In addition, we have begun compiling information available to date on genomics, proteomics, transcriptomics and population studies of consequence in the hope we can build on this in years to come., (Copyright © 2013 Australian Society for Parasitology Inc. All rights reserved.)

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p<0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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.

The genetic diversity within and among parasite populations provides clues to their evolutionary history. Here, we sought to determine whether mitochondrial and microsatellite DNA variation could be used to evaluate the extent of differentiation, gene flow and historical reproductive isolation among the freeze resistant parasites Trichinella nativa and the Trichinella T6 genotype infecting wolverines (Gulo gulo) in Nunavut, Canada. To this end, we genotyped Trichinella isolates derived from the diaphragms of 39 wolverines from this locale to reference strains of T. nativa and the Trichinella T6 genotype. Results showed that among a subset of 13 isolates examined, individuals resembled T. nativa in their mitochondrial DNA, but resembled the Trichinella T6 genotype when assayed at expansion segment V and the internal transcribed spacer of the nuclear rDNA. To adjudicate among these conflicting diagnoses, we further characterized each isolate at several nuclear microsatellite loci and again compared these to data from reference strains. Statistical assignment established that the nuclear genomes of most Nunavut isolates corresponded to those of the Trichinella T6 genotype; however, two isolates corresponded to T. nativa, and one isolate exhibited equal similarity to both reference strains. Taken as a whole, the evidence suggests that these isolates derive from the T. nativa matrilineage, but that their nuclear genomes resemble individuals previously designated as Trichinella T6. Assuming distinct lineages, this argues for cross-hybridization among these genotypes. Although introgression has occurred, recognizable genetic distinctions persist. One possibility is that selection disfavors the survival of hybrid offspring in most instances. Alternatively, the recent disappearance of glacial barriers might have increased contact, and therefore introgression. Broader geographic sampling will be required to determine the extent to which hybridization occurs beyond this particular geographic focus., (Published by Elsevier B.V.)

Incidence and mortality due to tuberculosis (TB) have been decreasing worldwide. Given that TB is a cosmopolitan disease, proper surveillance and evaluation are critical for controlling dissemination. Herein, mathematical modeling was performed in order to: 1) demonstrate a correlation between the incidence of TB in HIV-free patients in the US and Germany, and their corresponding mortality rates; 2) show the utility of the newly developed D-R algorithm for analyzing and predicting the incidence of TB in both countries; and 3) inform us on population death rates due to TB in HIV-negative patients. Using data published by the World Health Organization between 1990 and 2009, the relationship between incidence and mortality that could not be ascribed to HIV infection was evaluated. Using linear, quadratic and cubic curves, we found that a cubic function provided the best fit with the data in both the US (Y = 2.3588+2.2459X+61.1639X(2)-60.104X(3)) and Germany (Y = 1.9271+9.4967X+18.3824X(2)-10.350X(3)) where the correlation coefficient (R) between incidence and mortality was 0.995 and 0.993, respectively. Second, we demonstrated that fitted curves using the D-R model were equal to or better than those generated using the GM(1,1) algorithm as exemplified in the relative values for Sum of Squares of Error, Relative Standard Error, Mean Absolute Deviation, Average Relative Error, and Mean Absolute Percentage Error. Finally, future trends using both the D-R and the classic GM(1,1) models predicted a continued decline in infection and mortality rates of TB in HIV-negative patients rates extending to 2015 assuming no changes to diagnosis or treatment regimens are enacted.

Genome evolution studies for the phylum Nematoda have been limited by focusing on comparisons involving Caenorhabditis elegans. We report a draft genome sequence of Trichinella spiralis, a food-borne zoonotic parasite, which is the most common cause of human trichinellosis. This parasitic nematode is an extant member of a clade that diverged early in the evolution of the phylum, enabling identification of archetypical genes and molecular signatures exclusive to nematodes. We sequenced the 64-Mb nuclear genome, which is estimated to contain 15,808 protein-coding genes, at ∼35-fold coverage using whole-genome shotgun and hierarchal map-assisted sequencing. Comparative genome analyses support intrachromosomal rearrangements across the phylum, disproportionate numbers of protein family deaths over births in parasitic compared to a non-parasitic nematode and a preponderance of gene-loss and -gain events in nematodes relative to Drosophila melanogaster. This genome sequence and the identified pan-phylum characteristics will contribute to genome evolution studies of Nematoda as well as strategies to combat global parasites of humans, food animals and crops.

Incidences of H1N1 viral infections in Mainland China are collected by the Ministry of Health, the People's Republic of China. The number of confirmed cases and the timing of these outbreaks from May 13 to July 22, 2009 were obtained and subjected to a novel mathematical model to simulate the infection profile (time vs number). The model was predicated upon the grey prediction theory which allows assignment of future trends using limited numbers of data points. During the period of our analysis, the number of confirmed H1N1 cases in Mainland China increased from 1 to 1772. The efficiency of our model to simulate these data points was evaluated using Sum of squares of error (SSE), Relative standard error (RSE), Mean absolute deviation (MAD) and Average relative error (ARE). Results from these analyses were compared to similar calculations based upon the grey prediction algorithm. Using our equation, defined herein as equation D-R, results showed that SSE=6742.00, RSE=10.69, MAD=7.07, ARE=2.47% were all consistent with the D-R algorithm performing well in the estimation of future trends of H1N1 cases in Mainland China. Calculations using the grey theory had no predictive value [ARE for GM(1,1)=-104.63%]. To validate this algorithm, we performed a second analysis using new data obtained from cases reported to the WHO and CDC in the US between April 26 and June 8, 2009. In like manner, the model was equally predictive. The success of the D-R mathematical model suggests that it may have broader application to other viral infections among the human population in China and may be modified for application to other regions of the world., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: To assess the efficacy of the D-R model for defining trends in the appearance of Ceftazidime-resistant Escherichia coli., Methods: Actual data related to the manifestation of Ceftazidime-resistant E. coli spanning years 1996-2009 were collected from the China National Knowledge Internet. These data originated from 430 publications encompassing 1004 citations of resistance. The GM(1,1) and the novel D-R models were used to fit current data and from this, predict trends in the appearance of the drug-resistant phenotype. The results were evaluated by Relative Standard Error (RSE), Mean Absolute Deviation (MAD) and Mean Absolute Error (MAE)., Results: Results from the D-R model showed a rapid increase in the appearance of Ceftazidime-resistant E. coli in this region of the world. These results were considered accurate based upon the minor values calculated for RSE, MAD and MAE, and were equivalent to or better than those generated by the GM(1,1) model., Conclusion: The D-R model which was originally created to define trends in the transmission of swine viral diseases can be adapted to evaluating trends in the appearance of Ceftazidime-resistant E. coli. Using only a limited amount of data to initiate the study, our predictions closely mirrored the changes in drug resistance rates which showed a steady increase through 2005, a decrease between 2005 and 2008, and a dramatic inflection point and abrupt increase beginning in 2008. This is consistent with a resistance profile where changes in drug intervention temporarily delayed the upward trend in the appearance of the resistant phenotype; however, resistance quickly resumed its upward momentum in 2008 and this change was better predicted using the D-R model. Additional work is needed to determine if this pattern of "increase-control-increase" is indicative of Ceftazidime-resistant E. coli or can be generally ascribed to bacteria acquiring resistance to drugs in the absence of alternative intervention.

Four species of Cryptosporidium are routinely found in cattle: Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae, and Cryptosporidium andersoni. It is important to determine the species of Cryptosporidium in infected cattle because C. parvum is the only serious pathogen for humans as well as cattle. Identification of Cryptosporidium species and genotypes currently relies on molecular methods such as polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) or gene sequencing. Incorporation of these techniques in a routine veterinary diagnostic laboratory is cost prohibitive. As such, their applications are limited primarily to research and a few public health laboratories. To overcome this problem, a multiplex PCR assay was developed for simultaneously detecting the 4 species of Cryptosporidium that commonly infect cattle. This assay specifically identifies Cryptosporidium oocysts present in cattle feces, improves the detection of mixed infections, reduces the time and cost relative to current sequencing methods, and further demonstrates the shortcomings of sequencing as the definitive method for identification when analyzing samples containing mixed infections.

In 1990, the Human Genome Sequencing Project was established. This laid the ground work for an explosion of sequence data that has since followed. As a result of this effort, the first complete genome of an animal, Caenorhabditis elegans was published in 1998. The sequence of Drosophila melanogaster was made available in March, 2000 and in the following year, working drafts of the human genome were generated with the completed sequence (92%) being released in 2003. Recent advancements and next-generation technologies have made sequencing common place and have infiltrated every aspect of biological research, including parasitology. To date, sequencing of 32 apicomplexa and 24 nematode genomes are either in progress or near completion, and over 600k nematode EST and 200k apicomplexa EST submissions fill the databases. However, the winds have shifted and efforts are now refocusing on how best to store, mine and apply these data to problem solving. Herein we tend not to summarize existing X-omics datasets or present new technological advances that promise future benefits. Rather, the information to follow condenses up-to-date-applications of existing technologies to problem solving as it relates to parasite research. Advancements in non-parasite systems are also presented with the proviso that applications to parasite research are in the making.

Studying parasites of the genus Trichinella provides scientists of today many advantages. This is a group of zoonotic nematodes that circulates freely among wildlife hosts with one in particular, Trichinella spiralis that is exceptionally well adapted to domestic swine. Recent reports suggest that human infections from hunted animals are on the rise worldwide and numerous countries still experience problems with T. spiralis in their domestic food supplies. Trichinella is a genus whose members are easily propagated in the laboratories, have been used as models to investigate host-parasite relationships and parasitism among clade I organisms, and represent a poorly investigated link between the phylum Nematoda and other Metazoans. The importance of T. spiralis in better understanding the tree of life was so recognized that in 2004, its genome was carefully selected as one of only nine key non-mammalian organisms to be sequenced to completion. Since it was first discovered in 1835, this genus has expanded from being monospecific to eight species including four other genotypes of undetermined taxonomic rank. Inasmuch as discriminating morphological data have been scant, our understanding of the genus has been relegated to a compilation of molecular, biochemical and biological data. Herein, we provide a collection of information and up-to-date interpretations on the taxonomy, diagnostics, systematics, micro- and macroevolution, and the biogeographical and biohistorical reconstruction of the genus Trichinella.

Ostertagia ostertagi is a gastrointestinal parasitic nematode that affects cattle and leads to a loss of production. In this study, we present the first large-scale genomic survey of O. ostertagi by the analysis of expressed transcripts from three stages of the parasite: third-stage larvae, fourth-stage larvae and adult worms. Using an in silico approach, 2284 genes were identified from over 7000 expressed sequence tags and abundant transcripts were analyzed and characterized by their functional profile. Of the 2284 genes, 66% had similarity to other known or predicted genes while the rest were novel and potentially represent genes specific to the species and/or stages. Furthermore, a subset of the novel proteins were structurally annotated and assigned putative function based on orthologs in Caenorhabditis elegans and corresponding RNA interference phenotypes. Hence, over 70% of the genes were annotated using protein sequences, domains and pathway databases. Differentially expressed transcripts from the two larval stages and their functional profiles were also studied leading to a more detailed understanding of the parasite's life-cycle. The identified transcripts are a valuable resource for genomic studies of O. ostertagi and can facilitate the design of control strategies and vaccine programs.

Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90-99 and 148-161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC's were determined to be: 16-64 microg/ml against Escherichia coli; 32-128 microg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64-256 microg/ml against Pseudomonas aeruginosa. The MBC's were equivalent to or 1-fold greater than corresponding MIC's. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle.

Objective: To evaluate antimicrobial activity of bovine bactericidal permeability-increasing protein (bBPI)-derived synthetic peptides against mastitis-causing gram-negative bacteria., Sample Population: Bacterial isolates from the milk of cows with clinical mastitis., Procedures: 3 peptides were synthesized with sequences corresponding to amino acids 65 to 99 (bBPI(6,599)) or 142 to 169 (bBPI(142,169)) or the combination of amino acids 90 to 99 and 148 to 161 (bBPI(9,099, 148,161)) of bBPI. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these peptides against bacterial isolates from cows with mastitis were determined by use of a standardized broth microdilution assay. The ability of these peptides to retain their antimicrobial activity in serum and milk was also evaluated. Finally, bacterial lipopolysaccharide (LPS)-neutralizing activity of these peptides was assayed with the Limulus amebocyte lysate test., Results: Of the 3 peptides tested, bBPI(9,099, 148,161) had the widest spectrum of antimicrobial activity, with MIC and MBC values ranging from 16 to 64 Mg/mL against Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp and from 64 to 128 Mg/mL against Pseudomonas aeruginosa. None of the peptides had any growth-inhibitory effect on Serratia marcescens. The antimicrobial activity of bBPI(9,099, 148,161) was inhibited in milk, but preserved in serum. Finally, bBPI(142,169) and bBPI(9,099, 148,161) completely neutralized LPS., Conclusions and Clinical Relevance: bBPI(9,099, 148,161) is a potent neutralizer of the highly proinflammatory molecule bacterial LPS and has antimicrobial activity against a variety of gram-negative bacteria. The ability of bBPI(9099,148161) to retain antimicrobial activity in serum suggests a potential therapeutic application for this peptide in the management of gram-negative septicemia.

The diseases caused by parasitic nematodes in domestic and companion animals are major factors that decrease production and quality of the agricultural products. Methods available for the control of the parasitic nematode infections are mainly based on chemical treatment, non-chemical management practices, immune modulation and biological control. However, even with integrated pest management that frequently combines these approaches, the effective and long-lasting control strategies are hampered by the persistent exposure of host animals to environmental stages of parasites, the incomplete protective response of the host and acquisition of anthelmintic resistance by an increasing number of parasitic nematodes. Therefore, the challenges to improve control of parasitic nematode infections are multi-fold and no single category of information will meet them all. However, new information, such as nematode genomics, functional genomics and proteomics, can strengthen basic and applied biological research aimed to develop improvements. In this review we will, summarize existing control strategies of nematode infections and discuss ongoing developments in nematode genomics. Genomics approaches offer a growing and fundamental base of information, which when coupled with downstream functional genomics and proteomics can accelerate progress towards developing more efficient and sustainable control programs.

Human neurotrichinellosis is seldom reported. This is likely the result of the low incidence of parasites from the genus Trichinella in the United States domestic food supply, as well as difficulties in diagnosing the disease, especially when neither the organism nor the source of the infection are readily available. Although trichinellosis from domestic food supplies has been decreasing for many years, a resurgence has occurred in cases derived from the consumption of wild game. We report a rare case of neurotrichinellosis in the United States and implicate wild game as the source of the infection. These results suggest that clinicians should consider the potential for Trichinella infection in cases where wild game is common in the diets of the patients.

The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.

CD14 is a high-affinity receptor protein for the complex of bacterial LPS (LPS) and LPS binding protein in animals. Binding of the soluble form of CD14 (sCD14) to LPS, found in the outer membrane of Escherichia coli and other Gram-negative bacteria, enhances host innate immune responses, reduces the severity of mastitis, and facilitates clearance and neutralization of LPS, thus protecting against an excessive immune response to LPS and development of endotoxic shock. A truncated form of sCD14, carrying a histidine residue affinity tag for purification, was incorporated into Potato virus X for transient expression in Nicotiana benthamiana plants. Western blots probed with CD14-specific antibodies demonstrated that crude plant extracts and affinity-purified samples contained immunoreactive sCD14. Biological activity of plant-derived recombinant bovine sCD14 (PrbosCD14) was demonstrated in vitro by LPS-induced apoptosis and interleukin (IL) -8 production in bovine endothelial cells, and in vivo by enhancement of LPS-induced neutrophil recruitment. Finally, in PrbosCD14-infused glands subsequently infected with E. coli, lower numbers of viable bacteria were recovered and there was an absence of clinical symptoms, demonstrating prophylactic efficacy of PrbosCD14. This is the first report of a functionally active animal receptor protein made in plants and the prophylactic use of a plant-derived protein to reduce the severity of bacterial infections in animals.

A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.

Parelaphostrongylus tenuis is a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsis sp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence of P. tenuis from the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.

A non-isotopic single-strand conformation polymorphism ('cold' SSCP) technique has been assessed for the analysis of sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. Data are consistent with the ability of cold SSCP to identify intra-specific as well as inter-specific variability among Trichinella genotypes. The cold SSCP approach should be applicable to a range of other genetic markers for comparative studies of Trichinella populations globally.

There has been recent emphasis on developing better methods for detecting diseases of zoonotic and veterinary importance. This has been prompted by an increase in human disease agents detectable in environmental samples, the potential for bioterrorism, and the lowering of international trade barriers and expansion of personal travel, which are bringing previously considered exotic diseases to new geographical localities. To appreciate the complexities of developing detection methods and working with environmental samples, it is appropriate to review technologies currently in use, as well as those in development and presently limited to research laboratories. Discussion of parasite detection would not be possible without including methods for parasite sampling, concentration, and purification because it is often necessary to process large sample volumes prior to analysis, and no reliable methods are available for significantly amplifying parasites in vitro. Reviewing proven methods currently in use will provide a baseline for generating, accepting and implementing the more sensitive and specific methods under development today.

The largely unanticipated difficulties of parasite vaccine development have led us to a renewed awareness of the survival strategies evolutionarily embedded within parasites over hundreds of millions of years. We have grown to appreciate that efforts to disrupt parasite-host relationships are substantially compounded by our incomplete understanding of the complex immune responses that occur in the naturally infected host. Given the inability to transfer laboratory successes to field trials, research is leading us to conclude that genetically defined animal models may not be good predictors of the unique and disparate protective immune responses one can expect from the genetically heterogeneous populations of animals that represent the parasite's natural environment. This is further compounded by the abundance of mechanisms parasites have created for themselves to defend against immune intervention. Thus, in the never-ending saga of vaccine development, it is only appropriate that pitfalls and advancements be critiqued as they apply across parasite groups, with a look towards promising technologies that may propel this field to the level of scientific achievement once envisaged.

IL-4 and IL-13 are multi-functional cytokines with overlapping roles in the host defense against infection. Equally important in the regulation of IL-4 and IL-13 are their associated receptors. Though, their functional receptor complexes and signaling pathways are intricate and in some cases, share common elements, the specificity of the responses, nonetheless, resides in the structure and binding of the alpha-chain components. This report presents the cloning of the swine receptors IL-4Ralpha and IL-13Ralpha1 and the effects of parasite infection on their transcription. Pairwise alignment of predicted amino acid sequences indicates that the swine IL-13Ralpha1 is 86, 83, and 72% similar to canine, human and mouse sequences, respectively. Amino acid sequence conservation is appreciably lower between the swine IL-4Ralpha sequence and those from equine (72%), human (66%), and mouse (49%); however, noteworthy similarities were observed in their overall predicted secondary structures predominantly among the swine, equine, and human homologues. Relative levels of receptor mRNA in tissues from swine experimentally infected with the protozoan, Toxoplasma gondii (T. gondii) or the nematodes Ascaris suum (A. suum) or Trichuris suis (T. suis), which are known to induce Th1 or Th2 host responses, respectively, were measured by real-time PCR. Results indicated that within 14 days following infection, overall mRNA levels for IL-4Ralpha and IL-13Ralpha1 were elevated in T. gondii-infected animals and reduced in A. suum-infected animals. Levels of swIL-4Ralpha and swIL-13Ralpha1 mRNA in T. suis-infected animals varied coincidentally with the course of the infection and the location of the analyzed tissue.

A nonisotopic single-strand conformation polymorphism (SSCP) approach was employed to 'fingerprint' sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. In addition, phylogenetic analyses of the D3 sequence data set, employing three different tree-building algorithms, examined the relationships among all of them. These analyses showed strong support that the encapsulated species T. spiralis and T. nelsoni formed a group to the exclusion of the other encapsulated species T. britovi and its related genotypes Trichinella T8 and T9 and T. murrelli, and T. nativa and Trichinella T6, and strong support that T. nativa and Trichinella T6 grouped together. Also, these eight encapsulated members grouped to the exclusion of the nonencapsulated species T. papuae and T. zimbabwensis and the three representatives of T. pseudospiralis investigated. The findings showed that nonencapsulated species constitute a complex group which is distinct from the encapsulated species and supported the current hypothesis that encapsulated Trichinella group external to the nonencapsulated forms, in accordance with independent biological and biochemical data sets.

Objective: To characterize cytokine messenger RNA (mRNA) expression in intranasally vaccinated calves after bovine respiratory syncytial virus (BRSV) challenge., Animals: Twelve 8- to 12-week-old calves., Procedures: Calves received modified-live BRSV vaccine (vaccinated) or spent tissue culture medium (mock-vaccinated) intranasally, followed by challenge 30 days later with BRSV, or mock challenge with spent tissue culture medium (mock-challenge controls). Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA was measured in lungs, bronchoalveolar lavage (BAL) fluid cells, pharyngeal tonsils, and tracheobronchial lymph nodes, and tumor necrosis factor-alpha (TNF-alpha) mRNA was measured in lungs and BAL fluid cells by reverse transcriptase-competitive polymerase chain reaction assay., Results: Resistance to clinical signs of disease was conferred in vaccinated calves. Expression of TNF-alpha mRNA in lungs and BAL fluid cells was higher in mock-vaccinated calves than control or vaccinated calves. In the lung, IL-4 mRNA expression was higher in vaccinated calves than control or mock-vaccinated calves. In pharyngeal tonsils, expression of mRNA for IL-4 and IFN-gamma was higher in mock-vaccinated calves than control calves. In tracheobronchial lymph nodes, IFN-gamma mRNA expression was higher in mock-vaccinated calves than vaccinated calves., Conclusions and Clinical Relevance: Although vaccinated calves had decreased clinical signs of disease after BRSV challenge, compared with mock-vaccinated calves, this difference was not related to a T helper type 1 bias, as determined by increased expression of interferon-gamma mRNA relative to interleukin-4 mRNA in lungs, BAL fluid cells, or tracheobronchial lymph nodes of vaccinated calves. Pulmonary inflammation was decreased in vaccinated calves as determined by decreased expression of TNF-alpha mRNA.

Studies in mice and humans indicate that membrane CD14 (mCD14) on the surface of monocytes, macrophages and polymorphonuclear neutrophils (PMN) mediate activation of these cells by lipopolysaccharide (LPS). Soluble CD14 (sCD14), in the circulation, binds to LPS and blocks LPS binding to mCD14. The role of bovine CD14 in cellular activation by LPS is undefined. Changes in CD18 expression on PMN and steady state levels of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-8, sensitive markers for activation of leukocytes by LPS, were used to measure functional activity of recombinant bovine sCD14 (rbosCD14). Whole blood (n=3 cows) treated with LPS alone caused CD18 expression on PMN to increase by 12% (P<0.02), whereas pre-incubation of LPS with 10 or 100 microg/mL of rbosCD14 completely inhibited increase in CD18 expression. After treating whole blood with LPS at concentrations of 1, 100 or 10(4) ng/mL for 2 h, level of mRNA for TNF-alpha, IL-6 and IL-8 in leukocytes and concentration of TNF-alpha in plasma increased. However, pre-incubation of LPS with rbosCD14 inhibited the increase in TNF-alpha mRNA, but not the increase in IL-6 and IL-8 mRNA. Excess amount of anti-human CD14 monoclonal antibody (MAB) also inhibited LPS-induced increase in TNF-alpha mRNA. Preincubation of LPS with rbosCD14, or rbosCD14 plus MAB did not affect LPS-induced increase in TNF-alpha in plasma. Collectively, results indicate that rbosCD14 inhibit LPS-induced increase in CD18 expression and TNF-alpha mRNA. However, secretion of TNF-alpha was not inhibited by pre-incubation of LPS with rbosCD14. The TNF-alpha in plasma may partially induce transcription of IL-6 and IL-8, which contribute to the CD14-independent increase in level of mRNA for IL-6 and IL 8.

Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 micro g of total RNA from Trichinella spiralis newborn larvae. The library consisted of >125000 insert-containing clones. Approximately 40-50 x 10(3) clones were screened immunologically using sera from pigs experimentally infected with 7000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1716 bp sequence that was absent from the 1497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted.

Standard therapies including administration of potent antibiotics, aggressive fluid resuscitation and metabolic support have not been successful in relieving symptoms and reducing mortality associated with acute coliform mastitis. It is important to understand the pathophysiological response of the mammary gland to coliform infections when designing preventive or therapeutic regimens for controlling coliform mastitis. Our laboratory has previously shown that macrophages and polymorphonuclear neutrophils in milk express CD14 on their cell surface. In this study, we found that soluble CD14 (sCD14) is present in milk whey as a 46kDa protein reacted with anti-ovine CD14 antibody. Additional functional studies found that: (1) under serum-free condition, complexes of LPS-recombinant bovine soluble CD14 (rbosCD14) induced activation of mammary ductal epithelial cells (as measured by changes in interleukin-8 (IL-8) mRNA level by competitive RT-PCR) at low concentrations of LPS after 6 or 24h incubation (1-1000ng/ml), whereas LPS alone did not induce activation of mammary ductal epithelial cells at the same concentrations, and (2) intramammary injection of low concentrations of LPS did not increase concentration of leukocytes in milk. In contrast, LPS-rbosCD14 complex containing the same concentration of LPS increased the concentration of leukocytes in the injected mammary gland at 12 and 24h post-injection. These results indicate that rbosCD14 sensitizes mammary epithelial cells to low concentrations of LPS in vitro and in vivo. Endogenous sCD14 in milk may be important in initiating host responses to Gram-negative bacterial infections.