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Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.

Animals↗

Control of Leishmania major infection in mice lacking TNF receptors.

TNF participates in the induction of nitric oxide (NO) production and macrophage activation, leading to the elimination of intracellular pathogens. We previously found that TNF receptor p55-deficient mice (TNFRp55-/-) control replication of Leishmania major in vivo but fail to resolve their lesions. Here we report that mice lacking the p75 receptor (TNFRp75-/-) or both receptors (TNFRp55p75-/-), also control parasite replication, albeit mice lacking the p55 receptor (either TNFRp55-/- or TNFRp55p75-/-) are delayed in their elimination of L. major compared with controls. All TNF receptor-deficient mice developed a Thl-type immune response and up-regulated inducible NO synthase (iNOS) mRNA gene expression in lesions during infection. Thus, neither TNF receptor appears to be absolutely required for NO production or elimination of L. major in vivo. In vitro, however, while macrophages from naive TNFRp75-/- mice could be activated to produce NO and kill L. major, we observed a defect in NO production and parasite killing by resident peritoneal macrophages from naive TNFRp55-/- or TNFRp55p75-/- mice. However, when macrophages were elicited with leishmanial Ag from 4-wk-infected TNFRp55-/- or TNFRp55p75-/- mice, they produced NO and were leishmanicidal. These data suggest that the TNFRp75 plays no essential role in L. major infection in mice and that the p55 receptor may be required for optimal macrophage activation. However, the results also show that a mechanism exists by which macrophages can be primed in vivo during L. major infection to produce NO and kill L. major in the absence of signaling through either of the TNF receptors.

Animals↗

A DNA vaccine encoding CCL4/MIP-1beta enhances myocarditis in experimental Trypanosoma cruzi infection in rats.

Chagas' disease, caused by Trypanosoma cruzi, is a major cause of cardiovascular disease in Latin America. Exacerbated inflammation disproportional to parasite load characterizes chronic myocardial lesions in chagasic patients. Chemokines and their receptors are expected to account for the renewed inflammatory processes after the inoculation of the parasite, but their potential unique functions are far from being clear. Herein, we evaluated the effect of a DNA vaccine encoding CCL4/MIP-1beta, a CC-chemokine, in T. cruzi-elicited myocarditis in rats. Holtzman rats were given intramuscularly cardiotoxin and the CCL4/MIP-1beta DNA-containing plasmid (100microg) was delivered in this muscular site four times. Fourteen days after last immunization, animals were inoculated with a myotropical CL-Brener T. cruzi clone. Peak of parasitism was observed at day 15 after infection, preceding the peak of myocardial inflammation at day 20. Myocarditis was still intense at day 30, but the inflammatory infiltrates showed a more focal distribution. The expression of CCL2/MCP-1 and CCL4/MIP-1beta correlated closely with the kinetics of myocardial inflammation. The CCL4/MIP-1beta DNA vaccine induced an increase of the levels of the anti-CCL4/MIP-1beta observed in T. cruzi-infected animals. This was associated with an exacerbation of myocardial inflammation and fibrosis, although alterations in parasitemia and myocardial parasitism were not observed. Our data suggest that CCL4/MIP-1beta plays a role in preventing excessive inflammation and pathology rather than in controlling parasite replication.

Animals↗

Cambendazole for strongyle control in a pony band: selection of a drug-resistant population of small strongyles and teratologic implications.

Cambendazole (CBZ) treatments (20 mg/kg) given at 8-week intervals were used for parasite control in a breeding band of ponies (n = 33 to 43) during the period July 1974 to August 1978. Pre- and posttreatment worm egg counts on feces were used to evaluate efficacy of treatments after every 2nd treatment interval by monitoring changes in strongyle egg counts. Initially, effective reductions (expressed as 92% to 96% fewer worm eggs) were lessened to 70% by the 9th treatment (18 months) and to 28% by the 12th treatment, and thereafter, they fluctuated between 0% and 38%. Critical tests on animals culled during the 3rd year revealed that (i) large strongyles (Strongylus vulgaris and S edentatus) were efficaciously removed, (ii) 5 species of small strongyles (Cyathostomum catinatus, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus longibursatus, and Cylicostephanus minutus) exhibited some degree of resistance, and (iii) 11 species of small strongyles from 6 genera, including the 3 already named, were highly susceptible. Cylicostephanus minutus, in this population exhibited benzimidazole resistance for the first time, whereas Cylicostephanus goldi, previously recognized as resistant in other populations (B and C) in Kentucky, was susceptible to CBZ. A 2-fold increase in the drug-dose rate did not increase efficacy against the CBZ-resistant species of small strongyles, but a 4-fold increase small stronglyles, efficaciously removed 93% of the resistant small strongyles. Foaling rates in the brood band ranged between 38% and 61% for the 5 seasons. Three deformed foals occurred--1 in 1976 and 2 in 1977--among the total of 83 births. Present data did not evidence a probable week of early gestation for possible insult of the fetus by CBZ treatment.

Abnormalities, Drug-Induced↗

Helminth control used by trainers of thoroughbreds in England.

A telephone survey was conducted of the methods used to control parasitic worms at 106 thoroughbred training yards. Most of the horses were allowed access to grass and were therefore at risk of infection. The control methods relied primarily on the use of anthelmintics and appeared reasonably successful because only 44 per cent of trainers reported observing clinical signs of nematode infections. They dosed their horses frequently, 40 per cent treating every four to six weeks and 25 per cent treating every seven to eight weeks. New arrivals on yards were usually treated with anthelmintic but the strategies used would be unlikely to prevent the introduction of anthelmintic-resistant nematodes on most yards or to deal with encysted cyathostomes. Trainers were responsible for the design of most control schemes, and only 42 per cent of them based their choice of anthelmintic on veterinary advice.

Animals↗

Roles of free GPIs in amastigotes of Leishmania.

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.

Animals↗

Anthelmintic resistance in nematodes: extent, recent understanding and future directions for control and research.

Resistance has now been reported to all of the broad spectrum anthelmintic types currently available, namely to the benzimidazoles, levamisole/morantel and to ivermectin. The problem causes most concern for parasite control in sheep, but anthelmintic resistance has also been reported in nematodes of horses, goats, pigs and more recently cattle. Our understanding of the factors which select rapidly for resistance has increased and programmes of worm control which minimize selection for anthelmintic resistance are being developed and tested. One of the greatest problems encountered in attempting to reduce the selection for overt drug resistance is the need for more sensitive tests for developing resistance. In the long term, new approaches to chemotherapy and to overcoming anthelmintic resistance problems will arise from improving our understanding of the modes of action of, and mechanisms of resistance to, anthelmintics at the level of the receptor proteins and their genes.

Animals↗

Molecular characterization of an acetylcholinesterase implicated in the regulation of glucose scavenging by the parasite Schistosoma.

Acetylcholinesterase (AChE) present on the surface of the trematode blood fluke Schistosoma has been implicated in the regulation of glucose scavenging from the host blood. Determination of the molecular structure and functional characteristics of this molecule is a crucial first step in understanding the novel function for AChE and in evaluating the potential of schistosome AChE as a target of new parasite control methods. We have determined the primary structure of acetylcholinesterase from Schistosoma haematobium. Immunolocalization studies confirmed that the enzyme was present on the parasite surface as well as in the muscle. The derived amino acid sequence possesses features common to acetylcholinesterases: the catalytic triad, six cysteines that form three intramolecular disulphide bonds, and aromatic residues lining the catalytic gorge. An unusual feature is that the fully processed native enzyme exists as a glycoinositol phospholipid (GPI)-anchored dimer, but the sequence of the C?terminus does not conform to the current consensus for GPI modification. The enzyme expressed in Xenopus oocytes showed conventional substrate specificity and sensitivity to established inhibitors of AChE, although it is relatively insensitive to the peripheral site inhibitor propidium iodide. Distinctions between host and parasite AChEs will allow the rational design of schistosome-specific drugs and vaccines.

Acetylcholinesterase↗

Different patterns of the L-histidine decarboxylase (HDC) gene expression in mice resistant and susceptible to experimental cutaneous leishmaniasis.

OBJECTIVE AND DESIGN: In the present study the experimental murine Leishmania major ( L. major) infection model was used to investigate the role of histamine biosynthesis in cutaneous leishmaniasis. SUBJECTS, TREATMENT AND METHODS: A novel RNase Protection Assay (RPA) was developed and applied for the assessment of L-histidine decarboxylase (HDC) gene expression in organs of resistant C57BL/6 and susceptible BALB/c mice after infection with L. major. RESULTS: In the acute phase of infection a rapid but transient induction of HDC expression was observed in the infected lymph nodes of both strains correlating both temporally and spatially with parasite spread. The signal was present in the draining popliteal lymph nodes of both hosts, however, only susceptible mice known to be unable to control parasite dissemination showed induction of HDC in their distant periaortic lymph nodes as well. During the chronic phase of infection only the heavily parasitized organs of BALB/c mice showed high HDC gene expression. CONCLUSIONS: These data suggest that expression of the histamine-producing enzyme HDC in the decisive acute phase of leishmaniasis is not coupled with development of either appropriate Th1 or inadequate Th2 responses to L. major. We hypothesize, however, that during the chronic phase of infection elevated HDC levels, possibly of mast cell origin, are associated with Th2-dominated responses and serious disease development.

Acute Disease↗

Trypanosoma rhodesiense infection in mice: sex dependence of resistance.

There is large variation in the survival of inbred mouse strains infected with Trypanosoma rhodesiense (EATRO 1886). Of those strains that survived for at least 22 days postinfection, female mice were markedly more resistant than male mice. The longer a strain survived, the greater was the difference in survival between male and female mice. Parasite counts were higher in male mice than in females, suggesting that the decreased resistance of males was due to their relative inability to control parasite growth. To determine the possible role of an X-linked resistance gene, resistant (C57BL/6) and susceptible (BALB/c) mice were mated, and their F1 progeny were infected with T. rhodesiense. There was no difference in the resistance between reciprocal F1 male mice (C57BL/6 X BALB/c versus BALB/c X C57BL/6), indicating that an X-linked gene does not account for the difference in resistance between susceptible and resistant mice.

Animals↗

Seasonal translation of infective larvae of gastrointestinal nematodes of cattle and the effect of Duddingtonia flagrans: a 3-year pilot study.

A study was conducted over 3 years (1998-2000) to investigate larval availability of gastrointestinal nematodes from faeces of cattle reared under different parasite control schemes. These cattle were part of a parallel, but separate grazing trial, and were used as donor animals for the faecal material used in this experiment. At monthly intervals, faeces were collected and pooled from three groups of first-season grazing cattle. These groups were either untreated, ivermectin bolus treated or fed the nematophagous fungus Duddingtonia flagrans. The untreated and fungus treated animals were infected with gastrointestinal nematodes and the number of eggs per gram (epg) pooled faeces ranged between 50 and 700 in the untreated group and between 25 and 525 epg in the fungus treated group. Each year between June and September, artificial 1 kg dung pats were prepared and deposited on pasture and protected from birds. The same treatments, deposition times and locations were repeated throughout the study. Larval recovery from herbage of an entire circular area surrounding the dung pats was made in a sequential fashion. This was achieved by clipping samples in replicate 1/4 sectors around the dung pats 4, 6, 8 and 10 weeks after deposition. In addition, coinciding with the usual time of livestock turn-out in early May of the following year, grass samples were taken from a circular area centred where the dung pats had been located to estimate the number of overwintered larvae, which had not been harvested during the intensive grass sampling the previous year. It was found that recovery and number of infective larvae varied considerably within and between seasons. Although the faecal egg counts in 1999 never exceeded 300 epg of the faecal pats derived from the untreated animals, the abnormally dry conditions of this year generated the highest level of overwintered larvae found on herbage in early May 2000, for the 3 years of the study. Overall, biological control with D. flagrans significantly reduced larval availability on herbage, both during and between the grazing seasons, when compared with the untreated control. However, the fungus did not significantly reduce overwintered larvae derived from early season depositions (June and July), particularly when dung pats disappeared within 2 weeks after deposition. Very low number of larvae (<3 per kg dry herbage) were sporadically recovered from grass samples surrounding the ivermectin bolus faecal pats.

Animals↗

The impact of HIV-protease inhibitors on opportunistic parasites.

Opportunistic parasitic infections are an important cause of morbidity and mortality in people infected with HIV. Since the introduction of highly active antiretroviral therapy (HAART), there has been a marked reduction in the occurrence and clinical course of these parasitic infections. Although these changes have been attributed to the restoration of cell-mediated immunity induced by either non-nucleoside reverse transcriptase inhibitors or HIV protease inhibitors, in combination with at least two nucleoside reverse transcriptase inhibitors included in HAART, there is evidence that HIV protease inhibitors have a direct inhibitory effect on the proteases of parasites. The results of studies on opportunistic parasitic infections conducted both before and during the HAART era indicate the need to develop clinical trials on the efficacy of HIV protease inhibitors in controlling parasitic infections in individuals with HIV or other immunocompromised individuals and laboratory investigations on aspartyl proteases of parasites as an important target for the development of new drugs.

AIDS-Related Opportunistic Infections↗

Current trends in research into the waterborne parasite Giardia.

The waterborne flagellated parasite Giardia intestinalis continues to be the most frequent protozoan agent of intestinal disease world-wide, causing an estimated 2.8 x 10(8) cases per annum. Severe symptoms of diarrhea and sickness can be persistent and even life threatening in the immunocompromised, in infants, and in the aged, although self-limiting in the majority of patients. Despite a growing awareness and intensified research many uncertainties remain, especially with respect to the risk of potential zoonotic transmission. Water supplies can be monitored for cysts using automated cytofluorimetric immunoassays, but this does not measure infectivity. Filtration provides the best protection, because cysts are highly resistant to chlorine and ozone. Other incompletely elucidated aspects include mechanisms of pathogenicity, host reaction to infection, immunity and parasite control using vaccines or antigiardial compounds; the 5-nitroimidazole metronidazole is the most effective of these. Molecular typing of various isolates indicates that most animal parasites are not infective to humans, but those that are can be genotypically classified as assemblage A or B. The phylogeny of the organism remains uncertain, but there is a growing opinion that Giardia is not an ancient primitive eukaryote, but that it is derived from a more complex mitochondria-containing protozoon.

Animals↗

Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis.

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.

Animals↗

Tick control: thoughts on a research agenda.

Tick control is critical to the control of tick borne disease, while the direct impact of ticks on livestock productivity is also well known. For livestock, tick control today rests overwhelmingly on the twin approaches of genetics and chemical acaricides, although the disadvantages and limitations of both are recognized. The achievement of the full potential of vaccination, the application of biocontrol agents and the coordinated management of the existing technologies all pose challenging research problems. Progress in many areas has been steady over the last decade, while the acquisition of molecular information has now reached a revolutionary stage. This is likely to have immediate impact on the identification of potential antigens for improved vaccines and novel targets for acaricide action. In many circumstances, the rate limiting step in making scientific progress will remain unchanged, namely the resource constraint on evaluating these appropriately in large animals. For other approaches, such as the use of biocontrol agents, the limitation is likely to be less in the identification of suitable agents than in their delivery in an efficient and cost effective way. Our scientific understanding of the molecular basis for the tick vector-tick borne disease interaction is in its infancy but the area is both challenging and, in the long term, likely to be of great practical importance. What is arguably the most difficult problem of all remains: the translation of laboratory research into the extremely diverse parasite control requirements of farming systems in a way that is practically useful.

Animals↗

The influence of formalin, benzocaine and hyposalinity on the fecundity and viability of Polylabroides multispinosus (Monogenea: Microcotylidae) parasitic on the gills of Acanthopagrus australis (Pisces: Sparidae).

Adult Polylabroides multispinosus exposed in vivo to formalin (200 p.p.m. in sea water, 30 min) deposited a similar number of eggs in 24 h in vitro as did control parasites (sea water, 35 parts per thousand, p.p.t., salinity) and worms exposed in vivo to benzocaine (40 p.p.m. in sea water, 10 min). Worms laid more eggs at 30 p.p.t. salinity in vitro than at 20, 35, 10 and 5 p.p.t. salinity (in decreasing order). Formalin (200 p.p.m., 30 min) decreased the viability of recently laid eggs, the survival of oncomiracidia, removed 70% of juvenile and adult worms from the gills, but decreased only slightly the viability of eggs exposed in utero or after eyespots developed in vitro. Salinities below 30 p.p.t. reduced viability as did an increase in temperature from 24 to 28 degrees C. All adult and juvenile worms were removed by baths in formalin (400 p.p.m., 25 min) or fresh water (1 h).

Animals↗

A putative role for larval nematode infection in diarrhoeas of lambs which did not respond to anthelmintic drenches.

Attempts to control a summer diarrhoea in grazing Finnish landrace lambs which had been unresponsive to anthelmintics and coccidiostats were made by supplementing them with cupric oxide particles and withdrawing a magnesium-rich mineral, while maintaining parasite control measures. The diarrhoea persisted from July to September and plasma pepsinogen activities were raised, suggesting that the anthelmintic did not prevent abomasal damage; the jejunum of an affected lamb showed lesions of parasitic gastroenteritis. Small responses to cupric oxide particles and larger responses to the withdrawal of magnesium were deceptive, possibly being confounded by differences in parasite challenge. In another experiment Finnish landrace lambs were more susceptible to diarrhoea than Suffolk cross lambs in autumn. The susceptibility was then linked to a strong inhibition of worm egg output and may have been caused by a hypersensitive mucosal response to the larval challenge. Plasma pepsinogen concentrations were again raised in the Finnish landrace lambs and did not decline after treatment with anthelmintic, whereas the concentrations increased later in the Suffolk cross lambs, and were apparently responsive to anthelmintic. The cases of diarrhoea were similar to 'July disease' and may have been caused by continuous nematode infections which were only briefly controlled by drenches. Anthelmintic-unresponsive diarrhoea is the term proposed for the disorder, which may be controllable by devices releasing anthelmintic continuously or by a move to less infected pasture. Faecal egg counts remained low in the condition and were diagnostically misleading.

Animals↗