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Biomedical subjects

A J Trees

Publications and source records attributed to A J Trees.

At least 19 recordsLinked to original sources

Clinical aspects of 27 cases of neosporosis in dogs.

Twenty-seven cases of neosporosis in European dogs are described. The disease was confirmed by immunohistochemistry, electron microscopy, or a favourable response to treatment in the dogs with appropriate clinical signs, and by the presence of antibodies to Neospora caninum but not to Toxoplasma gondii. The affected dogs were two days to seven years old, and of 13 different breeds. Both sexes were affected and in most cases littermates remained normal. Twenty-one cases had an initial hindlimb paresis or ataxia, in which muscle atrophy was the most consistent clinical sign. Rigid hyperextension developed in approximately half of the cases. Anorexia and pyrexia were rare. Other clinical signs included forelimb ataxia, head tremors with tetraparesis and sudden collapse due to myocarditis. Titres of > or = 1:800 in the N caninum indirect fluorescent antibody test were detected in the 20 cases from which serum samples were taken. Such high titres are rare in healthy dogs and strongly suggest a diagnosis of neosporosis. Sixteen of the dogs received appropriate antiprotozoal treatment with clindamycin, potentiated sulphonamides and/or pyrimethamine; 10 made a full or functional recovery. Recovery was less likely in peracute cases with severe clinical signs, and when the treatment was delayed.

Animals

Use of excretory/secretory antigens for the serodiagnosis of Anoplocephala perfoliata cestodosis.

Whole worm extract (WWE) and excretory/secretory (E/S) antigens of Anoplocephala perfoliata were characterised by SDS-PAGE and their use in the serodiagnosis of equine cestodosis was evaluated. An enzyme-linked immunosorbent assay (ELISA) was used to compare WWE and E/S antigen as the capture layer in an antibody capture ELISA. E/S antigen gave the best differentiation between sera from tapeworm-positive and tapeworm-negative horses. The E/S-ELISA was optimised and validated against sera from horses of known tapeworm status. This assay gave a diagnostic sensitivity of 68% (n = 38) and a specificity in helminth-naive horses of 95% (n = 20). Western blot analysis of tapeworm-positive and tapeworm-negative sera revealed the immunoreactive proteins of E/S antigen to be those of 12 and 13 kDa.

Animals

In vitro responsiveness of Babesia bovis to imidocarb dipropionate and the selection of a drug-adapted line.

A South African stock of Babesia bovis was successfully resuscitated from liquid nitrogen, and cultured in microaerophilous stationary phase. The in vitro susceptibility of the B. bovis stock to titrated concentrations of imidocarb dipropionate was observed and the 50% inhibitory concentration (IC) was determined (8.7 x 10(7) g ml-1). A drug-adapted line was developed by culture in the presence of sub-inhibitory concentrations of imidocarb dipropionate and it had an IC50 eight times higher than that of its original stock (6.6 x 10(-6) g ml-1). The drug-adapted line was cryopreserved and resuscitated from liquid nitrogen. Continuous culture of the non-drug adapted line through 15 subcultures did not change the IC50 (8.3 x 10(-7) g ml-1).

Animals

Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell-free culture and from IFN gamma-activated macrophages.

The susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to H2O2 and NO either in cell-free culture or from IFN gamma-activated macrophages was examined. In cell-free culture, O. lienalis microfilariae were highly susceptible to H2O2 induced toxicity, exhibiting rapid reductions in motility and viability. The addition of exogenous catalase abrogated H2O2-induced killing. In contrast, B. malayi microfilariae were relatively resistant to H2O2, with concentrations as high as 50 microM having no effect on motility or viability. On exposure to NO, both species showed reductions in motility within 5-30 min, but longer was required to see effects on the viability of microfilariae. Parasites incubated with IFN gamma-activated macrophages also exhibited marked reductions in motility and viability. In cultures with B. malayi and activated macrophages, inhibition of these effects was achieved by the addition of either L-NMMA, to abolish NO production, or neutralizing anti-TNF alpha antibodies. Attempts to inhibit parasite killing by the addition of catalase to macrophage cultures were ineffective. The results of this study show that B. malayi and O. lienalis microfilariae have different susceptibility to H2O2, but are equally affected by exposure to NO. Moreover both species are killed by IFN gamma-activated macrophages and in the case of B. malayi, killing is dependent on the generation of NO via TNF alpha.

Animals

Characterization and immunoprotective properties of a monoclonal antibody against the major oocyst wall protein of Eimeria tenella.

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.

Animals

Effects of haemagglutination (lectin) inhibitory sugars on Theileria parva infection in Rhipicephalus appendiculatus.

We have previously described the presence of haemagglutinins in tissues of the tick, Rhipicephalus appendiculatus and determined their sugar specificities by inhibition experiments. In this study, haemagglutination inhibitory sugars are shown to have an effect in vivo on the abundance of Theileria parva infected salivary gland acini in Rhipicephalus appendiculatus. A significant increase (P < 0.05) was observed in T. parva acinar infection rates in the salivary glands of R. appendiculatus fed on ears of rabbits infused with melibiose and raffinose. In contrast, mannose and turanose (non-haemagglutination inhibitory sugars) did not cause elevation of T. parva acinar infection rates. The effect of melibiose in elevating acinar infections was observed when used only during T. parva maturation in the salivary glands but not during parasite pick-up from an infected bovine host. Stabilates produced from ticks with elevated acinar infections did not differ from control stabilates in infectivity to cattle, by comparison of prepatent periods to pyrexia, or parasitosis, or in the severity of reactions.

Animals

Interferon gamma inhibits the intracellular multiplication of Neospora caninum, as shown by incorporation of 3H uracil.

An assay was developed to quantify the growth of two different isolates of the protozoon Neospora caninum within ovine fibroblast cells in vitro by differential uptake of 3H uracil. The NC-1 isolate of N. caninum multiplied more quickly in culture than the NC Liverpool isolate, as reflected by increased incorporation of isotope by the former over a shorter period of time. After the parasites had left the ruptured host cells, there was very little incorporation of isotope. This suggested that multiplication occurred within and not outside the cells. Treatment of the cells with ovine recombinant interferon gamma for 24 h before infection significantly inhibited intracellular multiplication of the parasite.

Animals

Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer, Topper and Uggla).

Neospora caninum is an apicomplexan, protozoan parasite, which causes severe disease in dogs and cattle. It has previously been isolated only in the United States. A 5-week-old Boxer pup with a progressive hindlimb paresis was diagnosed as suffering from neosporosis on the basis of clinical signs and the presence of anti-Neospora antibodies in it, 2 litter-mates and its dam. Despite treatment with sulphonamides, the pup was euthanased 3 days later. The diagnosis of neosporosis was confirmed by immunohistochemical examination of muscle and CNS tissue sections from the pup. Parasites were isolated into Vero cell culture from the cerebrum, and confirmed as Neospora caninum by immunofluorescence with specific antibody, tachyzoite ultrastructure and 16S-like ribosomal RNA sequences. This isolate (designated NC-Liverpool) has been continuously passaged every 7-10 days. Its growth characteristics, ultrastructure and antigenic profile, as revealed by immunoblotting, have revealed no major differences from the American NC-1 isolate. Furthermore, no difference was seen when comparing the sequences of 16S-like ribosomal RNA and the ITS1 region of the two isolates.

Animals

Cellular and humoral immune responses to recombinant antigens in sheep infected with Toxoplasma gondii.

Immune responses to recombinant fragments of the Toxoplasma gondii antigens ROP2 and GRA2 were investigated in sheep naturally and experimentally infected with T. gondii oocysts. Specific serum antibodies to C-terminal fragments were detected by ELISA. Cell-mediated responses in peripheral blood mononuclear cells were demonstrated by proliferation and interferon-gamma production following in vitro stimulation with the ROP2 fragment. This data indicates the presence of epitopes for sheep B cells in the recombinant GRA2 fragment and for both B and T cells in the ROP2 fragment.

Animals

Evaluation of suramin, ivermectin and CGP 20376 in a new macrofilaricidal drug screen, Onchocerca ochengi in African cattle.

To aid the development of a macrofilaricidal agent for Onchocerca volvulus, the African bovine parasite, O. ochengi, was evaluated as a drug screen by testing three known filaricidal drugs. Groups of five Zebu cattle, naturally infected with more than 15 palpable O. ochengi nodules in the ventral skin, were treated with either suramin (10 mg/kg/day i.v. for 6 days), ivermectin (200 micrograms/kg, s.c.), CGP 20376 (20 mg/kg orally) or left untreated as controls and examined at intervals up to 137 days post-treatment (d.p.t.). After ivermectin treatment, microfilarial densities in the skin decreased within one week to virtually zero and remained at a very low level. A similar rapid and profound reduction was seen after CGP 20376 treatment, but by 137 d.p.t. microfilarial skin densities were approaching pre-treatment levels. With suramin, skin microfilarial densities fell to very low levels after 12 weeks but rose slightly by 137 d.p.t. Effects on the macrofilariae were assessed by sequential nodulectomies at -3 and 28, 84 and 137 d.p.t. By 137 d.p.t. embryogenesis was almost completely interrupted in the CGP 20376 and ivermectin treated animals, although not in the suramin treated group, but in all three groups the majority of remaining intrauterine microfilariae were pathologically altered. Degenerating intrauterine microfilariae accumulated in the ivermectin and in the CGP 20376, but not in the suramin treated worms. The motility of male and female worms was not reduced by any treatment except for female worms at 84 d.p.t. with CGP 20376. Viability of the worms as indicated by the MTT-formazan reduction assay was not reduced in any of the treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Africa

Serological evidence implicating Neospora species as a cause of abortion in British cattle.

By means of an immunofluorescence antibody test (IFAT), using in vitro cultured parasites as antigen, antibodies to Neospora species at titres > or = 1/1280 were found in 11 of 120 Scottish cattle that had recently aborted but in only one of 97 cattle from herds in which there had been no recent abortions (P < 0.01). The specificity of the antibodies was confirmed by the lack of cross reactivity between samples with high titres to Neospora and toxoplasma antigen in a direct agglutination test, and by the absence of reactivity at > or = 1/640 in the IFAT of convalescent sera from cattle infected experimentally with Toxoplasma gondii, Sarcocystis cruzi, Eimeria bovis, E alabamensis, Cryptosporidium parvum and Babesia divergens. These results demonstrate that Neospora species infection occurs commonly in aborting cattle in Britain, and that the IFAT may be a useful tool for investigating the infection.

Abortion, Veterinary

Aggregated oviposition in the Simulium damnosum complex is mediated by eggs in a laboratory bioassay.

The phenomenon of aggregated oviposition in blackflies (Diptera: Simuliidae) was investigated under controlled laboratory conditions, using wild-caught females of the Simulium damnosum complex in Sierra Leone. A method was developed for inducing Simulium females to lay eggs, and used as a bioassay to measure the responses of gravid S. damnosum s.l. to freshly laid eggs of the same species complex. In a series of two-choice tests, significantly more ovipositing flies chose substrates already containing eggs over control substrates (P = 0.004). The time from introduction of flies into the oviposition system to the onset of egg-laying was significantly less when eggs were already present (P = 0.049). Flies responded more quickly when more eggs were present and the relationship between egg-batch number and the time of this response was curvilinear (P = 0.012). Ecological advantages and disadvantages of such aggregation behaviour and the possible role of semiochemicals in its mediation are discussed.

Animals

Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen.

An indirect enzyme linked immunsorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from dogs is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen. A mixture of a monoclonal antibody to dog immunoglobulin G and a horse radish peroxidase conjugated antibody to mouse Ig was used to detect bound antibody. When the iscom preparation was analysed by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis it appeared to consist of a restricted number of proteins compared with whole parasite homogenates. In immunoblot analysis, using N. caninum positive sera from rabbits and dogs as probes, the major antigens recognized had approximate molecular weights between 30 and 45 and 17 to 19 kDa. Compared with an ELISA using a crude solubilized tachyzoite antigen, the iscom ELISA substantially improved the sensitivity and specificity (to 97.6% and 95.6%, respectively, against an immunofluorescence test, IFAT, as indicator of true status). There was a statistically significant positive correlation between IFAT titres and iscom ELISA OD450 values. The iscom ELISA absorbances (and the IFAT titres) of dogs with proven clinical infections were not higher than those from nonclinically affected, putatively infected dogs.

Animals