Transcriptional responses consistent with perturbation in dermo-epidermal homeostasis in septic sole ulceration.

The aim of this study was to evaluate transcriptional changes in sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis was removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis was obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time QPCR and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of pro-inflammatory transcripts interleukin 1 α (IL1A), interleukin1 ß (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), and caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A, interleukin 18 (IL18), tumor necrosis factor α (TNFA) and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration.

Molecular evidence sterile tissue damage during pathogenesis of pododermatitis aseptica hemorrhagica circumscripta is associated with disturbed epidermal-dermal homeostasis.

Podermatitis aseptica hemorrhagica circumscripta is associated with metalloproteinase 2 weakening of distal phalangeal suspensory structures and sinkage of the distal phalanx in the claw capsule. Pressure from the tuberculum flexorium on the sole epidermis and dermis produces hemorrhagic tissue injury and defective horn production appearing as yellow-red, softened claw horn in region 4 of the sole. A model of the MAPK/ERK signal cascade orchestrating epidermal-dermal homeostasis was employed to determine if sterile inflammatory responses are linked to disturbed signal transduction for epidermal homeostasis in sole epidermis and dermis. The objective was to assess shifts in target genes of inflammation, up- and downstream MAPK/ERK signal elements, and targeted genes supporting epidermal proliferation and differentiation. Sole epidermis and dermis was removed from lateral claws bearing lesions of podermatitis aseptica hemorrhagica circumscripta, medial claws from the same limb and lateral claws from completely normal limbs of multiparous, lactating Holstein cows. The abundance levels of targeted transcripts were evaluated by real-time QPCR. Lesion effects were assessed by ANOVA, and mean comparisons were performed with t-tests to assess variations between mean expression in ulcer-bearing or medial claw dermis and epidermis and completely normal lateral claw dermis and epidermis or between ulcer-bearing dermis and epidermis and medial claw dermis and epidermis. The lesions were sterile and showed losses across multiple growth factors, their receptors, several downstream AP1 transcription components, CMYC, multiple cell cycle and terminal differentiation elements conducted by MAPK/ERK signals and ß 4, α 6 and collagen 17A hemidesmosome components. These losses coincided with increased cytokeratin 6, ß 1 integrin, proinflammatory metalloproteinases 2 and 9, IL1B and physiologic inhibitors of IL1B, the decoy receptor and receptor antagonist. Medial claw epidermis and dermis from limbs with lateral claws bearing podermatitis aseptica hemorrhagica circumscripta showed reductions in upstream MAPK/ERK signal elements and downstream targets that paralleled those in hemorrhagic lesions. Inhibitors of IL1B increased in the absence of real increases in inflammatory targets in the medial claw dermis and epidermis. Losses across multiple signal path elements and downstream targets were associated with negative effects on targeted transcripts supporting claw horn production and wound repair across lesion-bearing lateral claws and lesion-free medial claw dermis and epidermis. It was unclear if the sterile inflammation was causative or a consequence of these perturbations.

A tale of three kingdoms: members of the Phylum Nematoda independently acquired the detoxifying enzyme cyanase through horizontal gene transfer from plants and bacteria.

Horizontal gene transfer (HGT) has played an important role in the evolution of nematodes. Among candidate genes, cyanase, which is typically found only in plants, bacteria and fungi, is present in more than 35 members of the Phylum Nematoda, but absent from free-living and clade V organisms. Phylogenetic analyses showed that the cyanases of clade I organisms Trichinella spp., Trichuris spp. and Soboliphyme baturini (Subclass: Dorylaimia) represent a well-supported monophyletic clade with plant cyanases. In contrast, all cyanases found within the Subclass Chromadoria which encompasses filarioids, ascaridoids and strongyloids are homologous to those of bacteria. Western blots exhibited typical multimeric forms of the native molecule in protein extracts of Trichinella spiralis muscle larvae, where immunohistochemical staining localized the protein to the worm hypodermis and underlying muscle. Recombinant Trichinella cyanase was bioactive where gene transcription profiles support functional activity in vivo. Results suggest that: (1) independent HGT in parasitic nematodes originated from different Kingdoms; (2) cyanase acquired an active role in the biology of extant Trichinella; (3) acquisition occurred more than 400 million years ago (MYA), prior to the divergence of the Trichinellida and Dioctophymatida, and (4) early, free-living ancestors of the genus Trichinella had an association with terrestrial plants.

Evolution and Biogeography of Haemonchus contortus: Linking Faunal Dynamics in Space and Time.

History is the foundation that informs about the nuances of faunal assembly that are essential in understanding the dynamic nature of the host-parasite interface. All of our knowledge begins and ends with evolution, ecology and biogeography, as these interacting facets determine the history of biodiverse systems. These components, relating to Haemonchus, can inform about the complex history of geographical distribution, host association and the intricacies of host-parasite associations that are played out in physiological and behavioural processes that influence the potential for disease and our capacity for effective control in a rapidly changing world. Origins and evolutionary diversification among species of the genus Haemonchus and Haemonchus contortus occurred in a complex crucible defined by shifts in environmental structure emerging from cycles of climate change and ecological perturbation during the late Tertiary and through the Quaternary. A history of sequential host colonization associated with waves of dispersal bringing assemblages of ungulates from Eurasia into Africa and processes emerging from ecosystems in collision and faunal turnover defined the arena for radiation among 12 recognized species of Haemonchus. Among congeners, the host range for H. contortus is exceptionally broad, including species among artiodactyls of 40 genera representing 5 families (and within 12 tribes of Bovidae). Broad host range is dramatically reflected in the degree to which translocation, introduction and invasion with host switching, has characterized an expanding distribution over time in North America, South America, southern Eurasia, Australia and New Zealand, coincidental with agriculture, husbandry and global colonization by human populations driven particularly by European exploration after the 1500s. African origins in xeric to mesic habitats of the African savannah suggest that historical constraints linked to ecological adaptations (tolerances and developmental thresholds defined by temperature and humidity for larval stages) will be substantial determinants in the potential outcomes for widespread geographical and host colonization which are predicted to unfold over the coming century. Insights about deeper evolutionary events, ecology and biogeography are critical as understanding history informs us about the possible range of responses in complex systems under new regimes of environmental forcing, especially, in this case, ecological perturbation linked to climate change. A deeper history of perturbation is relevant in understanding contemporary systems that are now strongly structured by events of invasion and colonization. The relaxation of abiotic and biotic controls on the occurrence of H. contortus, coincidental with inception and dissemination of anthelmintic resistance may be synergistic, serving to exacerbate challenges to control parasites or to limit the socioeconomic impacts of infection that can influence food security and availability. Studies of haemonchine nematodes contribute directly to an expanding model about the nature of diversity and the evolutionary trajectories for faunal assembly among complex host-parasite systems across considerable spatial and temporal scales.

The Identification of Haemonchus Species and Diagnosis of Haemonchosis.

Diagnosis is often equated with identification or detection when discussing parasitic diseases. Unfortunately, these are not necessarily mutually exclusive activities; diseases and infections are generally diagnosed and organisms are identified. Diagnosis is commonly predicated upon some clinical signs; in an effort to determine the causative agent, identification of genera and species is subsequently performed. Both identification and diagnosis play critical roles in managing an infection, and involve the interplay of direct and indirect methods of detection, particularly in light of the complex and expanding problem of drug-resistance in parasites. Accurate and authoritative identification that is cost- and time-effective, based on structural and molecular attributes of specimens, provides a foundation for defining parasite diversity and changing patterns of geographical distribution, host association and emergence of disease. Most techniques developed thus far have been grounded in assumptions based on strict host associations between Haemonchus contortus and small ruminants, that is, sheep and goats, and between Haemonchus placei and bovids. Current research and increasing empirical evidence of natural infections in the field demonstrates that this assumption misrepresents the host associations for these species of Haemonchus. Furthermore, the capacity of H. contortus to utilize a considerably broad spectrum of ungulate hosts is reflected in our understanding of the role of anthropogenic forcing, the 'breakdown' of ecological isolation, global introduction and host switching as determinants of distribution. Nuanced insights about distribution, host association and epidemiology have emerged over the past 30years, coincidently with the development of increasingly robust means for parasite identification. In this review and for the sake of argument, we would like to delineate the diagnosis of haemonchosis from the identification of the specific pathogen. As a foundation for exploring host and parasite biology, we will examine the evolution of methods for distinguishing H. contortus from other common gastrointestinal nematodes of agriculturally significant and free-ranging wild ruminants using morphological, molecular and/or immunological methods for studies at the species and genus levels.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Cessation of Trichinella spiralis transmission among scavenging mammals after the removal of infected pigs from a poorly managed farm: implications for trichinae transmission in the US.

Pigs infected with the zoonotic parasite Trichinella spiralis were detected on a farm in Maryland during an animal welfare investigation. Sera and/or tissues were collected from 49 pigs and three pig carcasses (7 weeks of age to adult, mixed sex). The tissues were tested for the presence of T. spiralis muscle larvae (ML) by tissue digestion, and the sera were tested for the presence of anti-Trichinella antibodies by ELISA. Seventeen of 50 (34%) pigs were infected with T. spiralis based on tissue digestion. Of these 17 pigs, sera were collected from 16; nine were serologically positive, three sera had OD values that were very close to the positive cut-off (0.30), but were still negative, and four were negative (suggesting that they had become infected within a few weeks of testing). All pigs that tested negative by tissue digestion for ML were also ELISA negative. The farm was subsequently depopulated of pigs. Six months later, testing of trapped scavenging mammals in the farm environment demonstrated that 41% were infected with T. spiralis. After 12 months, 10% of trapped animals were T. spiralis positive, and after 18 months, T. spiralis could not be detected in the scavenging mammal population surrounding the farm. Results of the study suggest that T. spiralis, typically transmitted in the peridomestic rat-pig-human cycle in the US, was not maintained in scavenging mammals in the absence of infected pigs.

Survival of North American genotypes of Trichinella in frozen pork.

North American genotypes of Trichinella spiralis (T-1), Trichinella nativa (T-2), Trichinella pseudospiralis (T-4), Trichinella murrelli (T-5), and Trichinella T-6 were examined for susceptibility to freezing in pork using time-temperature combinations that have been proven to inactivate T. spiralis. Infections were established in 3-month-old pigs of mixed sex and breed by oral inoculation of 10,000 muscle larvae (ML) (all genotypes, rodent-derived ML), 20,000 ML (T-1, T-4, and T-5; cat-derived ML), or 30,000 ML (T-2 and T-6; cat-derived ML). Pigs were euthanized 60 days postinoculation. Muscles from the tongue, masseter muscles, diaphragm, triceps, hams, neck, rump, and loins were ground, pooled, and mixed to ensure even distribution of larvae. Samples (20 g) containing each Trichinella species, genotype, and source combination were placed in heat-sealable pouches, transferred to a constant temperature refrigerant bath, and maintained according to defined time and temperature combinations. Larvae recovered from cold-treated pork samples were inoculated into mice to determine infectivity. Results indicated that the time-temperature combinations known to render pork safe for T. spiralis are sufficient to inactivate T. nativa and T-6 (the freeze-resistant isolates), T. murrelli (the most common sylvatic species in the United States excluding Alaska), and T. pseudospiralis (a species that lacks a muscle nurse cell). These data close a gap in knowledge about the effectiveness of freezing for inactivating these parasites in pork and should alleviate concern about the safety of frozen pork products from the United States.

Age, segment, and horn disease affect expression of cytokines, growth factors, and receptors in the epidermis and dermis of the bovine claw.

The aim of this study was to examine changes in RNA expression for growth factors, cytokines, and receptors in epidermal-dermal tissues of the bovine claw relative to host age, claw segment, and disease state of the horn. Epidermal-dermal tissues were collected from the coronary, wall, sole, and bulb segments of 8- to 9-mo-old Holstein fetuses, normal adult cows, and adult cows with sole ulceration. Anatomic and pathologic characteristics were determined in tissues stained with eosin and hematoxylin, and RNA expression levels were evaluated using real-time, quantitative PCR. In normal tissues, certain RNA expression levels were clearly affected by host age: 290.0-, 610.0-, 53.4-, and 8.1-fold greater expression of granulocyte-macrophage colony stimulating factor was observed in fetal coronary, wall, sole, and bulb segment relative to adult tissues, respectively. A claw segment effect was also observed in that IL-1alpha expression was greater (1.59-fold) in the normal adult wall relative to the coronary segment, and IL-18 expression was greater (16.2-fold) in the normal adult sole compared with the coronary segment and 2.88 greater in the fetal sole relative to the bulb segment. Sole ulceration was associated with hemorrhage, thrombosis, inflammation, and striking increases in IL-1beta, IL-18, and inducible nitric oxide synthase, and with less dramatic, albeit measurable, changes in IL-1 type I receptor, IL-1 receptor antagonist, and tumor necrosis factor-alpha. Amidst striking increases in keratinocyte growth factor receptor (i.e., 21.0-fold, 10.4-fold, 0, and 21.6-fold in the coronary, wall, sole, and bulb segments, respectively), a concomitant decrease occurred in keratinocyte growth factor (i.e., 0.80-, 0.54-, 0.56-, and 0.72-fold, respectively). The results demonstrated changes in disease state and, to a lesser extent, claw segment and were accompanied by alterations in the RNA expression of several cytokines, growth factors, and receptors present in the normal claw.

Bovine coronary region keratinocyte colony formation is supported by epidermal-dermal interactions.

Delineating the factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronary region keratinocytes and dermal fibroblasts, determine the colony-forming capacity of epidermal keratinocytes in the coronary region, and characterize transcriptional changes in specific cytokine, growth factor, and receptor genes during colony formation in coculture. Fibroblasts and keratinocytes from the coronary region of the lateral, hind limb claw were collected, and 5.0 x 10(3) and 7.5 x 10(3) keratinocytes were cultured in the presence or absence of fibroblast monolayers, respectively. The 2 densities of keratinocytes formed 144 +/- 15.8 and 183 +/- 26.9 colonies, respectively, in the presence of dermal fibroblasts, whereas no colonies developed in the absence of dermal fibroblasts. Keratinocytes with the ability to show colony formation comprised 1.09% +/- 0.16 to 1.77% +/- 0.28 of the keratinocyte population isolated from the coronary region. Keratinocyte-fibroblast cocultures developed a time-dependent increased expression of several growth factors, cytokines, and receptors. These findings demonstrated that keratinocytes from the bovine coronary region formed colonies in vitro and that colony formation occurred with an absolute dependence on dermal fibroblasts. Colony growth was associated with increased transcriptional expression of cytokine, growth factor, and receptor expression known to drive keratinocyte colony formation in other species. The results indicate that horn-producing keratinocytes must interact with dermal fibroblasts during normal tissue homeostasis in the bovine claw.

Integrating genomics and phylogenetics in understanding the history of Trichinella species.

In 2004, funding was received by Washington University's Genome Sequencing Center through NHGRI, to completely sequence several nematode genomes as part of a holistic effort to advance our understanding of the human genome and evolution within the Metazoa. Trichinella spiralis was among this group of worms because of its strategic location at the base of the phylum Nematoda, and the belief that extant species represented an ancient divergent event that occurred as early as the Paleozoic. At the same time, a concerted effort was put forth to solidify the phylogeny of extant species of Trichinella based upon molecular analyses of a multi-gene system to understand the history of the genus and thereby enhance utilization of the forthcoming sequence data. Since the inception of this research, several findings have emerged: (1) the size of T. spiralis genome estimated by flow cytometry (71.3 Mb) is substantially smaller than originally predicted (270 Mb); (2) to date, a subset of the total of 3,534,683 sequences have been assembled into a 59.3 Mb unique sequence; (3) 19% of the assembled sequence is comprised of repetitive elements; and (4) sequence data are predicated upon extant T. spiralis which probably diverged as little as 20 million years ago. Thus, the utility of the T. spiralis genome as representative of an archaic species must be tempered with the knowledge that encapsulated and non-encapsulated clades probably separated during the mid-Miocene as temperate ecosystems changed.

Trichinella murrelli in scavenging mammals from south-central Wisconsin, USA.

Tissues and serum from 59 raccoons (Procyon lotor), 42 coyotes (Canis latrans), and seven Striped Skunks (Mephitis mephitis) collected in Dane and Iowa Counties, Wisconsin, USA, between October 2005 and March 2006 were microscopically and serologically examined for the presence of Trichinella spp. Encapsulated larvae were found on compression slides prepared from tongue tissues from a few animals. Complete tissue digestion of tongues revealed that 19% of the raccoons, 26% of the coyotes, and none of the seven skunks tested were infected with Trichinella spp. Cats were subsequently experimentally infected by feeding them the raccoon tissues containing muscle larvae, and muscle larvae isolated from the collected tongues were experimentally transmitted to mice. Multiplex polymerase chain reaction analysis of the isolated muscle larvae demonstrated two distinct bands migrating at 127 base pairs (bp) and 316 bp in all samples, which together are diagnostic for Trichinella murrelli; the isolates were assigned Istituto Superiore di Sanita (ISS) codes ISS1656 through ISS1667, and ISS1708 through ISS1710 by the International Trichinella Reference Centre. These findings extend the geographic range of T. murrelli into Wisconsin, USA.

Developments and hurdles in generating vaccines for controlling helminth parasites of grazing ruminants.

As a direct consequence of rising drug resistance among common nematodes of grazing animals, efforts toward state-of-the-art vaccine development have clearly intensified in recent years, fuelled primarily by the advent of newer technologies in gene discovery, by advancements in antigen identification, characterisation and production. In this regard, it is appropriate to review progress that has been made in generating helminth vaccines and in particular, vaccines against common nematodes of production animals for consumption. In like manner, it is prudent to evaluate barriers that have hindered progress in the past and continue to present obstacles that must be solved when utilizing and depending on host immunity to attenuate parasitic infections.

Post-Miocene expansion, colonization, and host switching drove speciation among extant nematodes of the archaic genus Trichinella.

Parasitic nematodes of the genus Trichinella cause significant food-borne illness and occupy a unique evolutionary position at the base of the phylum Nematoda, unlike the free-living nematode Caenorhabditis elegans. Although the forthcoming genome sequence of Trichinella spiralis can provide invaluable comparative information about nematode biology, a basic framework for understanding the history of the genus Trichinella is needed to maximize its utility. We therefore developed the first robust and comprehensive analysis of the phylogeny and biogeographic history of Trichinella using the variation in three genes (nuclear small-subunit rDNA, and second internal transcribed spacer, mitochondrial large-subunit rDNA, and cytochrome oxidase I DNA) from all 11 recognized taxa. We conclude that (i) although Trichinellidae may have diverged from their closest extant relatives during the Paleozoic, all contemporary species of Trichinella diversified within the last 20 million years through geographic colonization and pervasive host switching among foraging guilds of obligate carnivores; (ii) mammalian carnivores disseminated encapsulated forms from Eurasia to Africa during the late Miocene and Pliocene, and to the Nearctic across the Bering Land Bridge during the Pliocene and Pleistocene, when crown species ultimately diversified; (iii) the greatest risk to human health is posed by those species retaining an ancestral capacity to parasitize a wide range of hosts; and (iv) early hominids may have first acquired Trichinella on the African savannah several million years before swine domestication as their diets shifted from herbivory to facultative carnivory.

A Trichinella murrelli infection in a domestic dog in the United States.

Trichinella murrelli infection was diagnosed in a naturally infected Beagle bitch from VA, USA, where encapsulated larvae were found in histological sections of several skeletal muscles. A laboratory reared dog fed infected muscles resulted in viable muscle larvae that were subsequently infective to Swiss-Webster mice. Multiplex PCR using larvae from the experimentally infected dog demonstrated two distinct bands migrating at 127 bp and 316 bp which together are diagnostic for T. murrelli; the isolate was assigned the ISS code: ISS1608 by the International Trichinella Reference Centre. This is the first report of T. murrelli infection in a companion animal.

Cytokine responses in immunized and non-immunized calves after Ostertagia ostertagi infection.

The objective of this study was to evaluate abomasal cytokine responses in helminth-naive calves and calves vaccinated with protective antigen fractions from Ostertagia ostertagi after an experimental challenge infection with infective third stage (L3) larvae. Abomasal lymph nodes and/or abomasal mucosa were collected and messenger RNA for the Th1 cytokines (IFN-gamma, IL-2, IL-12 p40 subunit), the Th2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-15) and the Th3/Tr cytokine TGF-beta was quantified by real-time RT-PCR. Vaccination had no effect on cytokine profiles in either the abomasal lymph nodes or the abomasal mucosa. However, following infection all calves showed a significant decrease in the Th1 cytokines, IFN-gamma and IL-12 p40, and a significant increase in the Th2 cytokines, IL-4, IL-5, IL-10 and IL-13 in the lymph nodes, compared to non-infected calves. No correlation between the Th2 response and protection induced by vaccination could be demonstrated. In contrast, a Th2 pattern was not observed in the mucosa of the infected calves, which exhibited an increase in IFN-gamma as well as in the Th2 cytokines IL-4, IL-5 and IL-10 mRNA. No significant association was observed in the abomasal mucosa between any examined cytokine mRNA level and immune effector responses such as parasite-specific antibodies or the number of mucosal mast cells or eosinophils.

Recent advances on the taxonomy, systematics and epidemiology of Trichinella.

Since Owen first described Trichinella as a human pathogen in 1835, the number of organisms comprising this genus has grown dramatically. Where it was once thought to be a monospecific group, this genus is now comprised of eight species and three additional genotypic variants that have yet to be taxonomically defined. Along with the growth in the genus and description of the parasites has come a concomitant increase in our understanding of the epidemiology and geographical distribution of these organisms. Recent expansion of the non-encapsulated group to include three species biologically defined by their unique host ranges encompassing mammals, birds and reptiles, has raised substantial questions as to the term, 'Trichinella-free' as it applies to geographical localities. A true appreciation of the adaptability of this genus to host and environmental selection factors, as well as its dissemination to the far reaches of the world can best be appreciated by reviewing what we know and what we hope to know about this ancient and elusive parasite. The review herein consolidates our current understanding of the taxonomy, epidemiology, and phylogeny of the genus Trichinella, and identifies areas where data are lacking and our knowledge requires additional clarification.

Trichinella pseudospiralis from a wild pig in Texas.

In December 2001, the routine inspection of a wild boar intended for human consumption revealed the presence of Trichinella ssp. larvae. Biological, morphological and genetic analyses demonstrated the parasite to be Trichinella pseudospiralis. This is the second report of T. pseudospiralis in the United States and the first report of the parasite in a food animal species in the U.S.

Trichinella nativa in a black bear from Plymouth, New Hampshire.

A suspected case of trichinellosis was identified in a single patient by the New Hampshire Public Health Laboratories in Concord, NH. The patient was thought to have become infected by consumption of muscle larvae (ML) in undercooked meat from a black bear killed in Plymouth, NH in October 2003 and stored frozen at -20 degrees C fro 4 months. In January 2004, a 600 g sample of the meat was thawed at 4 degrees C, digested in hydrochloric acid and pepsin, and larvae were collected by sedimentation. Intact, coiled, and motile ML were recovered (366 larvae per gram (l pg) of tissue), which were passed into mice and pigs. Multiplex PCR revealed a single 127 bp amplicon, indicative of Trichinella nativa. The Reproductive Capacity Index (RCI) for the T. nativa-Plymouth isolate in mice was 24.3. Worm burdens in the diaphragms of two 3-month-old pigs given 2,500 ML were 0.05 and 0.2l pg by 35 days post-inoculation, while 2.2 and 0.75 l pg were recovered from two 3-month-old pigs given 10,000 ML; no larvae were recovered from four 1-year-old pigs given 2,500 ML (n=2) or 10,000 ML (n=2). Viable larvae were also recovered from frozen black bear meat harvested at two additional locations, one in southern Ontario, Canada, and one in upstate New York, USA. Multiplex PCR using genomic DNA from these parasite samples demonstrated that both isolates were T. nativa. This is the first report of the freeze-resistant species, T. nativa, within the continental United States.