Oxygen-Mediated Enzymatic Polymerization of Thiol-Ene Hydrogels.

Materials that solidify in response to an initiation stimulus are currently utilized in several biomedical and surgical applications; however, their clinical adoption would be more widespread with improved physical properties and biocompatibility. One chemistry that is particularly promising is based on the thiol-ene addition reaction, a radical-mediated step-growth polymerization that is resistant to oxygen inhibition and thus is an excellent candidate for materials that polymerize upon exposure to aerobic conditions. Here, thiol-ene-based hydrogels are polymerized by exposing aqueous solutions of multi-functional thiol and allyl ether PEG monomers, in combination with enzymatic radical initiating systems, to air. An initiating system based on glucose oxidase, glucose, and Fe2+ is initially investigated where, in the presence of glucose, the glucose oxidase reduces oxygen to hydrogen peroxide which is then further reduced by Fe2+ to yield hydroxyl radicals capable of initiating thiol-ene polymerization. While this system is shown to effectively initiate polymerization after exposure to oxygen, the polymerization rate does not monotonically increase with raised Fe2+ concentration owing to inhibitory reactions that retard polymerization at higher Fe2+ concentrations. Conversely, replacing the Fe2+ with horseradish peroxidase affords an initiating system is that is not subject to the iron-mediated inhibitory reactions and enables increased polymerization rates to be attained.

Clinical efficacy and plasma concentrations of two formulations of buparvaquone in cattle infected with East Coast fever (Theileria parva infection).

East Coast fever, caused by the protozoan parasite Theileria parva, kills about 600,000 cattle annually in Africa. The hydroxynaphthoquinone compound buparvaquone (BPQ) is curative. Sixteen calves were infected with T. parva. On manifestation of disease symptoms, eight were injected with the original (pioneer) BPQ product and eight with a test product containing BPQ. All 16 calves were cured by one injection of 2.5 mg BPQ/kg bodyweight. The concentration of BPQ in blood plasma was monitored by HPLC. The mean observed C(max) of BPQ was 0.229 and 0.253 microg/mL of plasma, the mean observed time to reach this concentration (T(max)) was 2.62 and 2.12 h and the AUC (area under curve) was 4.785 and 4.156 microg h/mL, respectively, for the pioneer and test product. Considerable variations occurred in the plasma concentration of BPQ within each group. They showed no relationship with either clinical or parasitological parameters following treatment.

In vitro inhibition of Eimeria tenella invasion by indigenous chicken Lactobacillus species.

The aim of this study was to determine the effects of indigenous chicken Lactobacillus species isolates from different parts of the gastrointestinal tract on Eimeria tenella invasion in vitro and to characterise the nature of inhibition, if any. The effects of competitive exclusion, steric interference and bacterial extracellular factors on E. tenella invasion were examined in an MDBK cell model. Several Lactobacillus species were initially isolated from chickens and identified by biochemical characteristics and 16S-rRNA. All Lactobacillus species isolates tested, significantly inhibited E. tenella invasion. Steric interference did not affect parasite invasion. Extracellular metabolic factors secreted by Lactobacillus species isolates into the surrounding media were shown to inhibit parasite invasion and these factors appeared to be heat stable. These results show that the natural microflora of poultry can provide a source of E. tenella-inhibiting Lactobacillus species in vitro, and thus may contribute to the control of Eimeria infection.

Treatment of East Coast fever of cattle with a combination of parvaquone and frusemide.

Pulmonary oedema is a common sign of East Coast fever (ECF, Theileria parva infection) of cattle. A trial was conducted on farms in Uganda to compare a product containing both the antitheilerial compound parvaquone and the diuretic compound frusemide with one containing only parvaquone, in the treatment of ECF. The trial involved 40 clinical cases of ECF, some of them complicated by other infections, in cattle of all ages and on several farms. Confirmed cases were treated with either parvaquone+frusemide (P+F) or parvaquone alone (P). Survival after treatment with P+F was 77% compared with 71% with P. Five of the 10 fatalities were complicated cases. The cure rate for severe but uncomplicated ECF was 89% with P+F and 40% with P. Pulmonary signs were resolved within 24-48 h after treatment with P+F and clinical recovery was noticeably more rapid than with P. The antiparasitic effect of the two treatments was similar. P+F could be particularly useful when reporting, diagnosis or laboratory confirmation of ECF is delayed, because advanced cases are more likely to be encountered under these circumstances.

Treatment of East Coast fever: a comparison of parvaquone and buparvaquone.

Two trials were conducted to compare the efficacy of parvaquone and buparvaquone for the treatment of naturally acquired East Coast fever (ECF, Theileria parva infection) which, if untreated, is almost invariably fatal in European breeds of cattle. In the first trial 28 naive cattle were exposed in a paddock infested with ticks carrying a virulent form of the disease. Twelve were treated with each drug when they developed clinical ECF. All 24 cattle were cured. In the second study, 100 cases of ECF occurring naturally on farms in Kenya were treated, 50 with each drug. Parvaquone cured 44 (88%) buparvaquone cured 45 (90%). Intercurrent infections, predominantly anaplasmosis and bacterial pneumonia or scour, were treated specifically. It is concluded that parvaquone and buparvaquone are similarly effective in curing ECF and cure rates are maximised by accurate diagnosis and prompt treatment of both ECF and intercurrent infections.

Cost analysis of immunisation against east coast fever on smallholder dairy farms in Kenya.

A spreadsheet model was developed and used to estimate the total cost of immunising cattle against East Coast fever (ECF) based on the infection-and-treatment method. Using data from an immunisation trial carried out on 102 calves and yearlings on 64 farms in the Githunguri division, Kiambu district, Kenya, a reference base scenario of a mean herd of five animals, a 10% rate of reaction to immunisation and a 2-day interval monitoring regimen (a total of 10 farm visits) was simulated. Under these conditions, the mean cost of immunisation per animal was US$16.48 (Ksh 955.78); this was equivalent to US$82.39 (Ksh 4778.90) per five-animal farm. A commonly reported reactor rate of 3% would decrease the cost of US$14.63 (Ksh 848.29) per animal. Reducing the number of farm monitoring visits from 10 to 7 would reduce the total cost by 10%, justified if farmers are trained to undertake some of the monitoring work. The fixed costs were 53% of the total cost of immunisation per farm. The cost of immunisation decreased with increasing number of animals per farm, showing economies of scale.

A mild Theileria parva parasite with potential for immunisation against East Coast fever.

Twenty-three Friesian cattle were inoculated subcutaneously anterior to the left prescapular lymph node with 1 ml of a mild isolate of Theileria parva. The cattle developed low macroschizont parasitosis but no clinical reaction was observed. Thirty-five days later the cattle were grouped into five groups and challenged with five different Theileria parva isolates (four cattle-derived Theileria and one buffalo-derived Theileria). The cattle were all solidly immune to challenge with the cattle-derived Theileria isolates but three out of five of the cattle challenged with the buffalo-derived parasite died of theileriosis. All ten non-immunised control cattle developed severe theileriosis and were treated with buparvaquone (Butalex; Pitman-Moore).

Elimination of Theileria buffeli infections from cattle by concurrent treatment with buparvaquone and primaquine phosphate.

Attempts (some successful) were made to eliminate Theileria buffeli infections from 23 naturally or experimentally infected, splenectomised calves. The anti-theilerial hydroxynaphthoquinone derivative buparvaquone was used either alone or in combination with primaquine phosphate. After treatment the calves were monitored for infection for up to 26 weeks. Blood films were examined for piroplasms and serum antibody levels were measured using an immunofluorescence technique. Buparvaquone alone failed to eliminate infections. Infections were eliminated from 11 of 16 calves treated twice with buparvaquone and three or six times with primaquine phosphate. Theilerial parasites were not subsequently seen in these 11 calves nor were antibodies detected beyond the eighth week after treatment.

Effect of treatment of chronic theileriosis with buparvaquone on milk yields.

Chronic, undulating Theileria annulata infection was diagnosed as the cause of severely depressed milk yields in a herd of 44 Friesian cows in Egypt. The herd was divided at random into two groups of 22; one group was injected intramuscularly with a single dose of the experimental antitheilerial drug, buparvaquone, while the other remained untreated. Over the subsequent seven weeks milk yield in the treated group increased to double that of the untreated group (P less than 0.05). Pyrexia was controlled within two days of injection of buparvaquone and piroplasm parasitaemia was eliminated in one week. It is suggested that treatment of dairy cattle chronically infected with T. annulata using buparvaquone may have the dual beneficial effect of reducing the pathogenic effects of theileriosis, thereby permitting restoration of an impaired immune system, thus increasing resistance to other infections. If a similar effect could be produced in Bos indicus cattle in T. annulata endemic areas, treatment of indigenous cattle with buparvaquone could be a useful alternative to the introduction of Bos taurus blood as a way of boosting milk production.

Chemotherapy of East Coast fever: parvaquone treatment of Theileria parva parva at intervals after infection.

Groups of seven cattle were infected with Theileria parva stabilate and treated with parvaquone (20 mg kg-1 bodyweight) zero, four, eight, 12, 14 or 16 days after infection. Very early treatments resulted in a rapid recovery or no detectable parasitosis and some cattle were subsequently susceptible to homologous challenge. Treatments applied before extensive lymphoid or other organ damage had developed were successful and some cattle treated in advanced disease also recovered. Clinical pathological indications of liver or kidney damage were recorded very late in the disease suggesting that prompt diagnosis and treatment are more important than supportive therapy for survival. Treatment on day 8 after infection allowed the appearance of macroschizonts and a transient pancytopenia but no other disease signs. This group was solidly immune to challenge and this timing and treatment could be recommended for use in an infection and treatment method of immunisation.

Identification of drugs effective against Cowdria ruminantium using a mouse screening system.

Heartwater is a tick-transmitted rickettsial infection of ruminants, caused by Cowdria ruminantium. The tetracyclines are the only compounds available for therapy of the disease. A screen, using mice infected with C. ruminantium, was developed and used to identify new compounds with potential for the control of heartwater. A series of di-4-methyl-thiosemicarbazones was shown to be highly active in the mouse model and their efficacy was confirmed in further trials in sheep infected with C. ruminantium. The mouse screen was shown to be simple to operate and reliably predictive of activity against heartwater. Ways in which the screen may be improved are suggested.

Cowdria ruminantium infection in the mouse: a review.

The current knowledge of the pathogenicity, clinical signs and mortality of artificial infections by syringe inoculation of Cowdria ruminantium in laboratory and wild strains of mice is reviewed. It is concluded that a wide spectrum of pathogenicity for mice exists in stocks of the organism.

Efficacy, toxicity and metabolism of imidocarb dipropionate in the treatment of Babesia ovis infection in sheep.

An intramuscular dose of 1.2 mg kg-1 of imidocarb dipropionate (IMDP) was effective in controlling fatal infections with Babesia ovis in sheep. The sheep were infected by the intravenous injection of sheep blood containing B ovis. A severe recrudescence of infection occurred in most sheep 10 to 14 days after therapy but this could be controlled by a second dose of 1.2 mg kg-1 IMDP. Studies on the toxicology, residues and metabolism of IMDP showed this to be a safe dosage regimen. Transient or mild signs of toxicity were seen at 2.4 and 4.8 mg kg-1 IMDP. Severe toxicity was observed in sheep treated with 9.6 mg kg-1.

Treatment of Theileria annulata infection in calves with parvaquone.

Fifteen calves were infected by the injection of stabilate of a suspension of Hyalomma anatolicum anatolicum ticks infected with the Ankara strain of Theileria annulata. Three were kept untreated, as controls, and they all died of theileriosis. Three groups of four calves were treated intramuscularly with parvaquone (Clexon; Wellcome) when early signs of theileriosis were clinically apparent. One group received 20 mg (kg bodyweight)-1 of parvaquone 10 days after infection. Two of these calves were clinically cured and two died of theileriosis. The remaining two groups of four calves received two doses of parvaquone, each of 10 mg (kg bodyweight)-1, either on days 10 and 11 or days 10 and 12. Three calves in each group were clinically cured while one in each group died of theileriosis. Total parasitological cure was not achieved in any of the calves. No symptoms of toxicity due to parvaquone treatment were observed.

Antitheilerial activity of BW720C (buparvaquone): a comparison with parvaquone.

A series of hydroxynaphthoquinones, all derivatives of the antitheilerial hydroxynaphthoquinone parvaquone (993C, Clexon; Wellcome) was tested for antitheilerial activity against Theileria parva (Muguga) in vitro. BW720C (buparvaquone) was 20 times more active than parvaquone. When tested in vivo BW720C cured all 13 cattle infected with T parva and all six infected with T annulata treated at a dose rate of 2.5 mg (kg bodyweight)-1 while parvaquone at 20 mg kg-1 cured nine of 10 cattle. All 16 untreated control cattle died of theileriosis.

Novel anti-malarial hydroxynaphthoquinones with potent broad spectrum anti-protozoal activity.

Novel hydroxynaphthoquinones are reported with outstanding efficacy against Plasmodium, Eimeria and Theileria species. Biochemical evidence is presented for the selective toxicity of these compounds being due to inhibition of parasite respiratory systems.

Evaluation of a range of antimicrobial agents against the parasitic protozoa, Plasmodium falciparum, Babesia rodhaini and Theileria parva in vitro.

Eighteen antimicrobials commonly used in tissue culture were screened in three different protozoan test systems in order to establish their suitability for routine inclusion in protozoal cultivation systems. The human malaria parasite, Plasmodium falciparum, was inhibited by more than half the antibiotics tested at concentrations recommended for normal tissue culture use. Eight compounds were well tolerated and thus could be used prophylactically to prevent microbial contamination. These antimicrobials were the bactericidal aminoglycoside antibiotics, streptomycin, gentamicin and kanamycin, the bacteriostatic protein synthesis inhibitors, chloramphenicol and chlortetracycline and the antifungals, 5-fluorocytosine, nystatin and amphotericin B. Babesia rodhaini and Theileria parva were less sensitive than P. falciparum and tolerated all 18 compounds at concentrations well above 100 micrograms ml-1. Extension of the study to examine direct antiprotozoal action of these and other antimicrobials not normally used in culture confirmed that P. falciparum was significantly more sensitive than the other parasites. Tylosin, rifamycin, gramicidin D and valinomycin were all strongly antimalarial with IC50 values of 0.245, 1.20, 1.3 X 10(-3) and 1.9 X 10(-3) micrograms ml-1 respectively. This compares with a value of 1.35 X 10(-2) micrograms ml-1 for the standard antimalarial, chloroquine. Only valinomycin and, more particularly, gramicidin D were significantly active against B. rodhaini and T. parva. Gramicidin D was more effective, but more toxic, than the standard antiprotozoal agents tested at curing in vivo malarial and babesial infections in mice.

Virulence of two strains of Cowdria ruminantium in mice and their use to predict drug activity against heartwater.

A study was made of the infectivity of two mouse-adapted strains of Cowdria ruminantium in mice. The Kwanyanga strain was most virulent in Balb/C mice which died nine days after infection with homogenate of liver from infected mice. CD-1 mice were least susceptible of six strains tested. The du Plessis strain of C. ruminantium was equally virulent in all six mouse strains. The du Plessis strain in CD-1 mice was used as the basis of a drug screen to detect activity against heartwater (C. ruminantium infection) and was highly predictive when active compounds were tested in sheep infected with the Ball 3 strain of C. ruminantium.

Activity of 10 naphthoquinones, including parvaquone (993C) and menoctone, in cattle artificially infected with Theileria parva.

Ten naphthoquinones, including parvaquone (993C) (Clexon; Wellcome) and menoctone, were tested for activity in cattle artificially infected with Theileria parva, the causative organism of East Coast fever (ECF). Parvaquone cured all 14 cattle treated with a single dose of 20 mg/kg intramuscularly and all five treated twice with 10 mg/kg intramuscularly. Menoctone cured seven of 10 cattle treated with a single dose of 5 mg/kg intramuscularly. Of 25 untreated control cattle, 22 died of ECF. None of the remaining eight naphthoquinones was as active as parvaquone. Three esters of active naphthoquinones, designed as 'prodrugs' of their parent compounds, showed little or no activity in infected cattle despite being highly active in vitro against T parva. These results were instrumental in the selection of parvaquone for development as the first specifically active remedy for ECF.