Neoplastic mass in an experimental Mongolian gerbil.
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Animals↗
Biomedical subjects
T R Klei
Publications and source records attributed to T R Klei.
An experimental evaluation of methods used to enumerate mucosal cyathostome larvae in ponies.
With the increased interest in equine cyathostomes it has become apparent that some evaluations of methods currently used to count the various larval stages which occur in the mucosa would be beneficial. Experiments were conducted to investigate the effects of fixation and storage of mucosal tissues at -20 C on the accuracy of counting these larvae. The accuracy of counting developing larvae within the mucosa by transmural illumination (TMI) and by artificial digestion (DIG) of the mucosa was also compared. The data indicate that fixation of digested mucosa in PBS-buffered 5% or 10% formalin did not effect the enumeration of either early hypobiotic L3 or larger developing L3 or L4. Although not optimal, counting these larvae by either TMI of DIG after freezing did not significantly differ from counts made on fresh tissues. Significant differences were also not seen between counts of developing larvae made by TMI or DIG. Because DIG must be used to count EL3 and small developing L3, it is possible that TMI is not necessary in heavily infected equids.
Animals↗
Immunity in equine cyathostome infections.
Defining the characteristics of immunity and immune responses to equine cyathostome infections is clearly important to advancing our understanding of the development of these nematodes within the host, the clinical conditions attributed to them, and in developing more rational and novel strategies for their control. Nonetheless, little is currently known on this topic. Current data based on field observations, worm burdens and fecal egg counts suggest that horses acquire a resistance to cyathostome infection with age. This response is slow to develop and incomplete in that most horses regardless of age harbor significant populations of these nematodes. More convincing evidence has been obtained from experimental infections which indicate that mature horses previously exposed to infection are resistant to re-infection and this resistance is directed at all stages of the parasite life cycle. Further, some immunity against the developing stages within the mucosa appears to require less exposure and occurs in younger animals. Some non-specific events which induce expulsion of all species of lumenal dwelling nematodes also appear to take place post-infection with L3. Antibodies have been detected in limited studies against somatic extracts of adult worms. Not surprisingly, titers of these antibodies do not correlate resistance to re-infection. Serendipitous observations have, however, associated a greater expression of the gene for IL-4 with the spontaneous expulsion of lumenal parasites. The development of a usable model is required to further advance our knowledge in this area.
Animals↗
Mucosal larval recovery techniques of cyathostomes: can they be standardized?
The methods used currently for enumeration of mucosal stages of cyathostomes include transmural illumination (TMI) and peptic digestion (DIG). Enumerating the inhibited early L3 (EL3) and differentiating between mucosal developing L3 (DL3) and L4 (ML4) is only possible when DIG is used. However, in some studies higher numbers of DL (DL3 + ML4) have been found when using TMI as opposed to DIG. This finding, however, is not consistent. Moreover, results are not consistent when DIG and TMI are compared for treated and control groups of horses in drug trials. Studies performed on the effect of freezing on DIG and TMI also show inconsistent results between laboratories. However, there is, evidence that digested samples can be preserved in 5-10% formalin or in 70% ethanol when used as a buffered isotonic solution. Based on these results it is premature to recommend standardized mucosal larval recovery techniques. Consequently, TMI and DIG should both be applied. Recommendations for further research for the development of standardized techniques are given.
Animals↗
Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves.
Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes sequences identical to wild-type for the generation of standard curves, is technically facile, less reagent-and labor-intensive than competitive methods, easily accommodates high sample throughput without the use of radioactive labels, and generates replicate samples to allow statistical analysis of the data. We demonstrate the utility of the assay, which is easily adapted to any cloned mRNA sequence, using equine interleukin-10 (IL-10). Both IL-10 and beta-actin cDNA were amplified in triplicate PCR reactions from oligo-dT primed RT reactions. Dilutions of plasmid DNA encoding the respective sequence, equine IL-10 or beta-actin, were also amplified in triplicate reactions in the same run. Beta-actin cycling parameters were modified to maintain the amplification in its exponential phase by decreasing both cycle number and cDNA volume relative to the parameters used for cytokine amplification. Following amplification, aliquots of the PCR reactions were hybridized with sequence-specific tris (2,2'-bipyridine) ruthenium II chelate labeled oligonucleotide probes and quantified using the QPCR System 5000. Plasmid derived values were used to generate a standard curve for the interpolation of mRNA content in unknown cDNA samples. Beta-actin values were used to derive a factor for the relative normalization of differences among cDNA samples that are inherent in the RNA extraction and RT steps. This assay resolves at least 2-fold differences in message, is reproducible, and has a dynamic range on the order of 3 logs.
Actins↗
Workshop on irradiated larval vaccines.
The development of anthelmintic resistance in gastrointestinal parasites of sheep and goats has meant that there is an urgent need for non-chemical control of these parasites. In order to investigate a potential alternative method of control, a workshop on irradiated larval vaccines (ILV) was held in conjunction with the Second International Conference on Novel Approaches to the Control of Helminth Parasites of Livestock. The objectives of the workshop were to: (1) Review knowledge of irradiated helminth vaccines through the presentation of invited and contributed papers. (2) Identify opportunities for an ILV and in which host/parasite/production systems. (3) Determine what are the gaps in knowledge required to produce and apply an ILV. The workshop concluded that ILVs could be useful as research tools in the further definition of immune responses to parasites and in stimulating the immune response in young sheep and goats to facilitate the estimating of heritability of faecal egg counts. Although the workshop identified some problems associated with a live attenuated vaccine, an ILV could be a useful control measure in the production systems identified by the workshop. Before an ILV could be used on farms, additional research is required into the period of protection afforded by an ILV especially under field conditions. The efficacy of an ILV could be improved by interaction between host genotype and nutritional status and is deserving of further investigation.
Animals↗
Vaccination against Strongylus vulgaris in ponies: comparison of the humoral and cytokine responses of vaccinates and nonvaccinates.
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Animals↗
Transglutaminase activity in equine strongyles and its potential role in growth and development.
Transglutaminases (E.C. 2.3.3.13) are a family of Ca(2+)-dependent enzymes that stabilize protein structure by catalyzing the formation of isopeptide bonds. A novel form of transglutaminase has been identified and characterized that seem to play an important role in growth, development, and molting in adult and larval stages of filarial nematodes. The aim of this study was to identify the ubiquitous nature of this enzyme in other nematodes and to measure its significance to larval growth, molting, and development. For this purpose, equine Strongylus spp. were used. Activity of this enzyme was identified in extracts of larvae and adults of Strongylus vulgaris, S. edentatus, Parascaris equorum and Cylicocyclus insigne. The significance of transglutaminase in the early growth and development of Strongylus vulgaris, S. edentatus and S. equinus was tested by adding specific inhibitors, monodansylcadaverine (MDC) or cystamine (CS), to in vitro cultures of third (L3) and fourth stage larvae (L4). The viability, molting and growth of these nematode species were affected by both inhibitors. Cystamine promoted abnormal development of Strongylus edentatus L3, resulting in an aberrant expansion of the anterior end. Addition of these inhibitors to cultures of L4 also reduced growth of the three species. The results indicated that transglutaminase is present in a wide array of nematode parasites and may be important in growth and development of their larval stages.
Animals↗
Immunity in experimental murine filariasis: roles of T and B cells revisited.
We have reevaluated the contributions of T and B cells in Brugia malayi infection by utilizing knockout mice on a uniform background (C57BL/6J). We find that B-cell-deficient mice are more permissive to infection than T-cell-deficient mice.
Animals↗
Granulomatous inflammatory response to recombinant filarial proteins of Brugia species.
The lymphatic inflammatory response in Brugia-infected jirds peaks early during primary infections and then decreases in severity as judged by the numbers of lymph thrombi present within these vessels. Antigen-specific hypersensitivity reactions in these animals was measured by a pulmonary granulomatous inflammatory response (PGRN) induced by somatic adult worm antigen (SAWA)-coated beads, and by cellular proliferative responses of renal lymph node cells. The kinetics of these responses temporally correspond to lymphatic lesion formation. The importance of any single antigen to the induction of this inflammatory response has not been elucidated. In this study, the PGRN was used to measure the cellular immune response to four recombinant filarial proteins during the course of a primary B. pahangi infection. These proteins were BpL4, glycoprotein (glutathione peroxidase) gp29, heat shock protein (hsp) 70, and filarial chitinase. All were fusion proteins of maltose-binding protein (MBP). Control beads included those coated with diethanolamine (DEA), SAWA, or MBP. The measurements of PRGN were made at 14, 28, 56, and > 150 days postinfection (PI) in infected jirds, in jirds sensitized with SAWA, and in uninfected jirds. The secretory homolog of glutathione peroxidase gp29 was the only recombinant protein tested that induced a significantly greater PGRN (P < 0.05) than controls. This was seen at 28 days PI. These observations indicate that gp29 may be part of the worm antigen complex that induces an early inflammatory response, a response similar to that observed with SAWA. These studies indicate that this approach is useful in investigating the functional ability of specific proteins in the induction and down-regulation of immune-mediated inflammatory responses elicited by filarial parasites. Absence of a granulomatous response to the other recombinant proteins used may be related to the nature and sensitivity of the assay used or the character of recombinant proteins tested.
Adult↗
Host NK cells are required for the growth of the human filarial parasite Brugia malayi in mice.
Human lymphatic filariasis, which afflicts an estimated 120 million people worldwide, is caused by the large nematode parasites Wuchereria bancrofti and Brugia malayi. Filarial nematodes require both an arthropod vector and a mammalian host to complete their life cycle. Within the definitive (mammalian) host, the lymphatic filarial parasites reside in the lymph nodes and lymphatics, a seemingly hostile environment for infectious agents, since the location exposes them to the immune defenses of the host. We present data here that suggest that the growth of B. malayi in the mammalian host is dependent on host NK cell function. Comparisons of worm survival and development in different strains of mice with varying levels of NK cell activity reveal that NOD/LtSz-scid/scid and NOD/LtSz-scid/scid B2m(null) mice (with diminished to absent NK cell activity respectively), are nonpermissive to worm growth, while C.B-17-scid/scid mice with normal NK cell activity are highly permissive. Depletion of NK cells in the permissive C57BL/6J-scid/scid mice renders them nonpermissive to B. malayi growth, whereas stimulation of NK cells in NOD/LtSz-scid/scid mice makes them permissive. Tg epsilon26 mice, which lack NK and T cells, are nonpermissive, but, when reconstituted with NK cells by adoptive transfer of bone marrow cells from C57BL16J-scid/scid mice, are rendered permissive. This requirement for NK cell activity may explain the site specificity of these parasites. Furthermore, these data suggest that the interaction of the host immune system with the filarial parasite is double edged, with both host protective and parasite growth-promoting activities emanating from the former.
Animals↗
Experimental cyathostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: comparison of parasite burdens, immunity and colonic pathology.
Eighteen mixed-breed, naturally infected ponies ranging in age from 1 to 16 yr and four cyathostome-naïve ponies reared and maintained under parasite-free conditions ranging in age from 1 to 4 yr were used in this study. Naturally-infected ponies were treated with 1 dose of ivermectin (IVM) at 200 micrograms kg-1, followed by a 5-day regimen of oxibendazole (OBZ) at 20 mg kg-1 to remove existing cyathostome burdens; cyathostome-naïve control ponies were treated with IVM alone. The naturally infected ponies were matched on age and gender, then randomly assigned to one of three treatment groups of six animals per group; the four cyathostome-naïve ponies constituted a fourth group. Following OBZ treatment, Group 1 ponies were treated with pyrantel tartrate (PT) in their pelleted ration; the remaining ponies received only the pelleted ration. Beginning on experiment Day 3, a daily challenge infection of 10(4) mixed cyathostome larvae was administered orally to ponies of Group 1, Group 2 and the cyathostome-naïve controls. Group 3 ponies served as unchallenged controls to determine residual parasite burdens following IVM/OBZ treatment. Necropsy examinations were performed on three Group 3 ponies on Day 1; the remainder of the necropsy examinations began on Day 41. Cyathostome burdens were evaluated by recovery of larvae and adults from the luminal contents, by digestions of the intestinal mucosa, and by mural transillumination of full-thickness intestinal sections. Differences in postchallenge clinical responses were also compared. Necropsy examinations included comparisons of grossly visible inflammation of the large bowel, weights of biopsy specimens from each region, and histologic evaluations of these biopsies. Parasite recoveries at necropsy indicated a strong protective effect derived from daily PT treatment. Mean weights of intestinal biopsies corresponded with worm burdens, but histological evaluation did not reveal architectural or cellular changes to account for the increase in weight; therefore, edema was suspected. A strong age-related resistance to challenge infection was apparent in both the PT-treated and control groups by virtue of the lower mean worm burdens found in older ponies compared to younger ponies of the same treatment group; however, daily PT treatment of older ponies reduced the variability of their worm burdens to a uniformly low level. Comparisons of luminal and mucosal parasite burdens of age stratified nontreated controls further suggest that the age related resistance, which is acquired, targets increasing numbers of parasite stages as this resistance matures. Further, there is no evidence for an immune mediated acquisition of hypobiotic L3.
Age Factors↗
Down regulation of macrophage activation in Brugia pahangi-infected jirds (Meriones unguiculatus).
The macrophage is a major component of the inflammatory response induced by lymphatic tissue-dwelling filariae. Intraperitoneal (i.p.) infections with Brugia pahangi in Mongolian gerbils, or jirds (Meriones unguiculatus), induce a peritoneal inflammatory response characterized by accumulation of numerous macrophages and fewer eosinophils. This inflammatory response is associated with the release of microfilariae by female worms. The aim of this study was to investigate the activation state of the peritoneal macrophages during the course of i.p. infections with either male or female worms. Activation was determined by a toxoplasmacidal assay and assays which measured the production of tumor necrosis factor (TNF)-like activity and nitric oxide (NO) production. The development of these assays with jirds was initially conducted in parallel with the mouse system, which served as a positive control. Jird macrophages became activated to kill Toxoplasma gondii by in vivo immunization with Mycobacterium bovis BCG in a pattern similar to that of mouse macrophages. However, unlike the mouse system, supernatants from purified protein derivative- or concanavalin A-stimulated jird splenocytes plus lipopolysaccharide failed to activate jird macrophages in vitro or induce NO production. These results indicate that factors involved in jird macrophage activation may differ from those demonstrated in the mouse system and other systems. i.p. infections of 15 days in duration with either male or female worms induced macrophage activation as measured by Toxoplasma killing and TNF production. These responses decreased as the infection progressed to the chronic period on a time course that parallels the down regulation of experimental B. pahangi granulomas. There was no evidence of NO production by activated jird macrophages. These data indicate that macrophage function is down modulated during filarial infection and suggest that mechanisms involved in macrophage deactivation are related to those that induce down modulation of the systemic granulomatous inflammatory response in the jird. This response is not dependent on the microfilarial stage of the parasite and is also independent of mechanisms which induce peritoneal accumulations of macrophages.
Animals↗
Hypoxanthine phosphoribosyltransferase cDNA in gerbils (Meriones unguiculatus).
Hypoxanthine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes conversion of hypoxanthine and guanine to their respective mononucleotides. Because of its ubiquitous nature, HPRT is known as a "housekeeping" gene and has been frequently used as an internal control in reverse transcriptase-polymerase chain reaction (RT-PCR) quantification of cytokine mRNA. Cloning and sequencing of the gerbil HPRT cDNA sequence is an important step in the development of RT-PCR procedures in this model. Two forms of gerbil HPRT cDNA were isolated and molecularly characterized.
Amino Acid Sequence↗
Efficacy of moxidectin 2% oral gel against second- and third-instar Gasterophilus intestinalis De Geer.
In a trial designed to evaluate the efficacy of the recommended dosage of moxidectin 2% oral gel against the gastric stages of Gasterophilus spp., 14 ponies were selected from a herd on the basis of the inclusion criterion of the presence of Gasterophilus spp. eggs attached to their hair coats. After random allocation, the ponies were treated with 1 of 2 treatments, moxidectin 2% equine gel in a single dose at the commercial dosage of 400 microg moxidectin/kg body weight or placebo gel. The animals were necropsied 14 days posttreatment. Efficacies against second- and third-instar Gasterophilus intestinalis De Geer were 100% and 99.5%, respectively.
Administration, Oral↗
Foals raised on pasture with or without daily pyrantel tartrate feed additive: comparison of parasite burdens and host responses following experimental challenge with large and small strongyle larvae.
Three groups of foals were raised under different management programs in this study: Group 1 (n = 6) and Group 2 (n = 6) were raised with their dams on pasture; Group 3 foals (n = 5) were raised under parasite-free conditions. Mares and foals of Group 1 received daily pyrantel tartrate (PT) treatment with their pelleted feed ration, whereas mares and foals of Groups 2 and 3 received only the pelleted ration. Pasture-reared foals were weaned and moved to a heavily contaminated pasture for 5 weeks. Group 1 foals continued to receive daily PT treatment whereas Group 2 foals received only the pelleted feed ration. Following this period, all foals were moved into box stalls. Half of each group was challenged with 10(3) Strongylus vulgaris infective third-stage larvae (L3), 5 x 10(3) Strongylus edentatus L3 and 10(5) mixed cyathostome L3; the remaining half served as unchallenged controls. Necropsy examinations were performed 6-week post-challenge for evaluation of parasite burdens and lesions. Daily PT treatment of Group 1 reduced the patent cyathostome infections of both mares and foals and was effective in reducing pasture burdens of infective larvae. Daily treatment of Group 1 foals during weaning continued to suppress EPG levels; however, it did not prevent large strongyle infections during the weaning period. Group 1 foals were more sensitive to challenge than Group 2 foals, which did not exhibit any post-challenge disturbances. Group 1 foals were equally susceptible to challenge as parasite-free foals.
Animal Feed↗
Brugia pahangi: differential induction and regulation of jird inflammatory responses by life-cycle stages.
It has been hypothesized that different life-cycle stages of filarial nematodes induce different host responses. This concept was examined in the Brugia pahangi-jird model of lymphatic filariasis by measuring the kinetics of inflammatory responses to parasite antigens following intraperitoneal inoculation of different life-cycle stages. For this purpose, viable female or male worms, L3, L4, or microfilarial stage, were used. Dead worms served as controls. Worm and microfilarial burdens, pulmonary granulomatous inflammation (PGRN) to soluble adult worm antigen (SAWA)-coated beads, and peritoneal eosinophil and macrophage numbers were assessed at different days post-inoculation. All jirds inoculated with any of these life-cycle stages developed an early PGRN to SAWA which was later significantly reduced. Only viable worms induced down-regulation of the PGRN response. These results indicate that the hyporesponsive state is induced and maintained by all life-cycle stages. Also, the degree of granulomatous response was influenced by worm burden, with larger worm burdens inducing lower initial levels of PGRN to SAWA. Peritoneal inflammatory responses differed from the systemic response in that numbers of macrophages increased with time and microfilarial accumulation. No correlation was observed between peritoneal inflammatory responses measured by eosinophil and macrophage numbers and PGRN to SAWA.
Animals↗
Immunological control of gastrointestinal nematode infections.
Control of nematode parasitism by an active manipulation of the host immune response has been a goal of veterinary and medical parasitologists for decades. The reality of achieving this goal has been questioned vigorously and demonstrations of the feasibility of using immunological control under field conditions are minimal. Nevertheless, with the rapid growth of modern biotechnology and the identification of novel parasite molecules as vaccine targets, the potential for success in this area has recently generated considerable excitement. The induction and regulation of the ruminant immune response against nematode parasites can be controlled either by management programs which include anthelmintic treatment or by vaccination. Both approaches will be discussed in this session.
Animals↗