ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Biological Evolution , Carbon-Nitrogen Lyases/analysis , Gene Transfer, Horizontal , Helminth Proteins/analysis , Nematoda/genetics , Animals , Bacteria/genetics , Plants/geneticsABSTRACT
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.
Subject(s)
Biological Evolution , Haemonchus/physiology , Host-Parasite Interactions , Animal Distribution , Animals , Animals, Domestic/parasitology , Biodiversity , Climate , Ecosystem , Haemonchus/classification , Host Specificity , Humans , Phylogeography , Time FactorsABSTRACT
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.
Subject(s)
Goat Diseases/parasitology , Haemonchiasis/veterinary , Haemonchus/isolation & purification , Sheep Diseases/parasitology , Animals , Goat Diseases/diagnosis , Goats , Haemonchiasis/diagnosis , Haemonchiasis/parasitology , Haemonchus/classification , Haemonchus/genetics , Haemonchus/immunology , Ruminants , Sheep , Sheep Diseases/diagnosis , Sheep Diseases/epidemiologyABSTRACT
We present a minimal one-dimensional deterministic continuous dynamical system that exhibits chaotic behavior and complex transport properties. Our model is an overdamped rocking ratchet with finite dissipation, that is periodically kicked with a δ function driving force, without finite inertia terms or temporal or spatial stochastic forces. To our knowledge this is the simplest model reported in the literature for a ratchet, with this complex behavior. We develop an analytical approach that predicts many key features of the system, such as current reversals, as well as the presence of chaotic behavior and bifurcation. Our analytical approach allows us to study the transition from regular to chaotic motion as well as a tangent bifurcation associated with this transition. We show that our approach can be easily extended to other types of periodic driving forces. The square wave is shown as an example.
ABSTRACT
Black bears (Ursus americanus) are hosts for two important zoonotic parasites, Toxoplasma gondii and Trichinella spp. and bears are hunted for human consumption in the USA. Little is known of the genetic diversity of T. gondii circulating in wildlife. In the present study, antibodies to T. gondii were found in juice from tongues of 17 (25.7%) of 66 wild black bear from Maryland during the hunting season of 2010 and 2011. Antibodies to T. gondii were assessed by the modified agglutination test. Tongues of 17 seropositive bears were bioassayed in mice and viable T. gondii was isolated from three samples. These three T. gondii isolates (TgBbMd1-3) were further propagated in cell culture and DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Results revealed three genotypes. TgBbMd1 is a Type 12 strain (ToxoDB PCR-RFLP genotype #4) and TgBbMd2 is ToxoDB PCR-RFLP genotype #216, and TgBbMd3 is a Type II clonal strain (ToxoDB PCR-RFLP genotype #1). The isolate TgBbMd2 was highly virulent for outbred Swiss Webster mice; all infected mice died of acute toxoplasmosis. Results indicate that mouse virulent strains of T. gondii are circulating in wildlife in the USA. These 66 tongues in addition to tongues collected during hunts in previous years were further investigated for the presence of muscle larvae of Trichinella spp. Tongues from 40 bears in 2005, 41 in 2006, 51 in 2007, 56 in 2008, 68 in 2009, 67 in 2010, and 66 in 2011 were subjected to digestion with pepsin/HCl and microscopic examination. Two bears were infected with Trichinella spp.; one in 2008 and one in 2009. Genotyping of collected muscle larvae revealed that the infecting species in both cases was Trichinella murrelli.
Subject(s)
Toxoplasma/genetics , Toxoplasmosis, Animal/parasitology , Trichinella/genetics , Trichinellosis/veterinary , Ursidae , Animals , Female , Genotype , Male , Maryland/epidemiology , Mice , Tongue/parasitology , Toxoplasma/classification , Toxoplasmosis, Animal/epidemiology , Trichinellosis/epidemiology , Trichinellosis/parasitologyABSTRACT
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.
Subject(s)
Apyrase/immunology , Apyrase/metabolism , Calcium/metabolism , Trichostrongyloidea/enzymology , Trichostrongyloidea/immunology , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Antigens, Helminth/genetics , Apyrase/genetics , Cations, Divalent/metabolism , DNA, Complementary/genetics , DNA, Helminth/genetics , Enzyme Activators/metabolism , Gene Expression Profiling , Helminth Proteins/genetics , Molecular Sequence Data , Ostertagia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sheep , Sheep Diseases/immunology , Trichostrongyloidea/genetics , Trichostrongyloidiasis/immunology , Trichostrongyloidiasis/veterinaryABSTRACT
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.
Subject(s)
Apyrase/classification , Calcium/pharmacology , Nucleotidases/metabolism , Ostertagia/enzymology , Ostertagia/growth & development , Animals , Bedbugs/enzymology , Blotting, Western , Esophagus/enzymology , Gene Library , Helminth Proteins/classification , Helminth Proteins/metabolism , Immunohistochemistry , Larva/enzymology , Nucleotidases/classification , Salivary Glands/enzymologyABSTRACT
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.
Subject(s)
Opossums/parasitology , Raccoons/parasitology , Swine Diseases/parasitology , Trichinella spiralis/isolation & purification , Trichinellosis/transmission , Trichinellosis/veterinary , Animal Husbandry , Animals , Animals, Wild/parasitology , Antibodies, Helminth/blood , Antigens, Helminth/isolation & purification , Communicable Diseases/transmission , Disease Reservoirs , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Humans , Maryland/epidemiology , Prevalence , Swine , Swine Diseases/epidemiology , Swine Diseases/prevention & control , Swine Diseases/transmission , Trichinellosis/epidemiology , Trichinellosis/parasitology , Trichinellosis/prevention & controlABSTRACT
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.
Subject(s)
Freezing , Genotype , Meat/parasitology , Trichinella/classification , Trichinella/genetics , Animals , Cat Diseases/parasitology , Cats , Food Preservation , Mice , North America , Swine , Swine Diseases/parasitology , Trichinellosis/parasitology , Trichinellosis/veterinaryABSTRACT
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.
Subject(s)
Cytokines/metabolism , Foot Diseases/veterinary , Gene Expression Regulation , Hoof and Claw/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Receptors, Growth Factor/metabolism , Age Factors , Animals , Cattle , Cattle Diseases/metabolism , Dermis/metabolism , Epidermis/metabolism , Female , Foot Diseases/metabolism , Hoof and Claw/pathologyABSTRACT
A deterministic overdamped ratchet driven by a periodic square driving force is shown to display chaotic behavior. The system has neither temporal nor quenched noise but the strong nonlinearity of the driving force produces a very rich bifurcation pattern with synchronized as well as chaotic regions. This pattern disappears if a sinusoidal force replaces the square force. This unexpected behavior is explained by decomposing the system into two exactly solvable subsystems, each with its own characteristic transit time, so that the ratio between the period of the driving force and the transit times can be analyzed. The transition from synchronized to chaotic motion can be explained by means of a one-dimensional Poincaré map. Our results can be experimentally confirmed in a number of systems, including the three-junction superconducting quantum interference devices ratchet, the rocking ratchet effect for cold atoms, and the Josephson vortex ratchet.
ABSTRACT
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.
Subject(s)
Cattle/growth & development , Cattle/metabolism , Dermis/metabolism , Epidermis/metabolism , Gene Expression Regulation, Developmental , Hoof and Claw/cytology , Animals , Cattle/genetics , Coculture Techniques , Cytokines/genetics , Female , Fibroblasts/cytology , Gene Expression Profiling , Intercellular Signaling Peptides and Proteins/genetics , Keratinocytes/cytology , Keratinocytes/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Genome, Helminth , Genomics , Phylogeny , Trichinella/genetics , Animals , Female , Flow Cytometry , Male , Trichinella/classificationABSTRACT
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.
Subject(s)
Coyotes/parasitology , Mephitidae/parasitology , Raccoons/parasitology , Trichinellosis/veterinary , Animals , Animals, Wild , Female , Food Chain , Genetic Markers , Male , Polymerase Chain Reaction/veterinary , Trichinella/growth & development , Trichinella/isolation & purification , Trichinellosis/epidemiology , Trichinellosis/parasitology , Wisconsin/epidemiologyABSTRACT
A trapping mechanism is observed and proposed as the origin of the anomalous behavior recently discovered in transport properties of overdamped ratchets subject to an external oscillatory drive in the presence of quenched noise. In particular, this mechanism is shown to appear whenever the quenched disorder strength is greater than a threshold value. The minimum disorder strength required for the existence of traps is determined by studying the trap structure in a disorder configuration space. An approximation to the trapping probability density function in a disordered region of finite length included in an otherwise perfect ratchet lattice is obtained. The mean velocity of the particles and the diffusion coefficient are found to have a nonmonotonic dependence on the quenched noise strength due to the presence of the traps.
ABSTRACT
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.
Subject(s)
Animals, Domestic/parasitology , Helminthiasis, Animal/prevention & control , Helminths/immunology , Intestinal Diseases, Parasitic/veterinary , Ruminants/parasitology , Vaccines , Animals , Animals, Domestic/genetics , Antigens, Helminth/genetics , Antigens, Helminth/immunology , Drug Resistance , Genetic Variation , Helminthiasis, Animal/immunology , Helminths/drug effects , Helminths/genetics , Host-Parasite Interactions , Intestinal Diseases, Parasitic/immunology , Intestinal Diseases, Parasitic/prevention & control , Ruminants/genetics , Vaccines/immunologyABSTRACT
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.
Subject(s)
Biological Evolution , Trichinella/genetics , Animals , Humans , Models, Genetic , Molecular Sequence Data , Sequence Alignment , Trichinellosis/parasitologyABSTRACT
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.