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.

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.

*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.

Divergence at mitochondrial and ribosomal loci indicates the split between Asian and European populations of Trichinella spiralis occurred prior to swine domestication.

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.

Gastrointestinal parasites of a reintroduced semi-wild plains bison (Bison bison bison) herd: Examining effects of demographic variation, deworming treatments, and management strategy.

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).

Wild ruminants as reservoirs of domestic livestock gastrointestinal nematodes.

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.

The transcriptomes of the cattle parasitic nematode Ostertagia ostartagi.

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.

Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties.

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 (Treg 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.

Bovine CD14 receptor produced in plants reduces severity of intramammary bacterial infection.

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 probable case of human neurotrichinellosis in the United States.

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.

A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis.

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.

Parasitic nematodes - from genomes to control.

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.

Antimicrobial activity of bovine bactericidal permeability-increasing protein-derived peptides against gram-negative bacteria isolated from the milk of cows with clinical mastitis.

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.

Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.

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.