Host ABC transporter proteins may influence the efficacy of ivermectin and possibly have broader implications for the development of resistance in parasitic nematodes.

ABC transporter proteins function to extrude compounds from the cell. These proteins present an obstacle for treatment and for overcoming drug resistance as they are expressed by both host and parasite, and function similarly. The contribution of host ABC proteins to drug efficacy was examined using ivermectin and a Brugia malayi model system. Parallel in vitro and in vivo experiments were conducted using equal concentrations of ivermectin. The motilities and fecundity of B. malayi exposed to ivermectin in vitro were significantly lower than those treated in vivo. The higher motilities were correlated with low concentrations of ivermectin in worms extracted from treated hosts. The expression of ABC proteins was significantly higher in worms treated in vitro compared to those treated in vivo as well as in gerbils treated with ivermectin than in non-treated controls. The results suggest that host ABC transporters may influence the efficacy of ivermectin.

ABC transporters influence sensitivity of Brugia malayi to moxidectin and have potential roles in drug resistance.

Some ABC transporters play a significant role in human health and illness because they confer multidrug resistance (MDR) through their overexpression. Compounds that inhibit the drug efflux mechanism can improve efficacy or reverse resistance. Of the eight described ABC transporter subfamilies, those proteins conferring MDR in humans are in subfamilies A, B, C, and G. In nematodes, transporters in subfamilies B and C are suggested to confer resistance to ivermectin. The Brugia malayi ABC transporter superfamily was examined to assess their potential to influence sensitivity to moxidectin. There was an increase in expression of ABC transporters in subfamilies A, B, C, and G following treatment. Co-administration of moxidectin with inhibitors of ABC transporter function did not enhance sensitivity to moxidectin in males; however, sensitivity was significantly enhanced in females and microfilariae. The work suggests that ABC transporters influence sensitivity to moxidectin and have a potential role in drug resistance.

Inventory and analysis of ATP-binding cassette (ABC) systems in Brugia malayi.

ABC systems are one of the largest described protein superfamilies. These systems have a domain organization that may contain 1 or more transmembrane domains (ABC_TM1F) and 1 or 2 ATP-binding domains (ABC_2). The functions (e.g., import, export and DNA repair) of these proteins distinguish the 3 classes of ABC systems. Mining and PCR-based cloning were used to identify 33 putative ABC systems from the Brugia malayi genome. There were 31 class 2 genes, commonly called ABC transporters, and 2 class 3 genes. The ABC transporters were divided into subfamilies. Three belonged to subfamily A, 16 to subfamily B, 5 to subfamily C, 1 to subfamily E and 3 to subfamilies F and G, respectively. None were placed in subfamilies D and H. Similar to other ABC systems, the ABC_2 domain of B. malayi genes was conserved and contained the Walker A and B motifs, the signature sequence/linker region and the switch region with the conserved histidine. The ABC_TM1F domain was less conserved. The relative abundance of ABC systems was quantified using real-time reverse transcription PCR and was significantly higher in female adults of B. malayi than in males and microfilaria, particularly those in subfamilies B and C, which are associated with drug resistance.

Brugia malayi: in vitro effects of ivermectin and moxidectin on adults and microfilariae.

The effect of ivermectin and moxidectin on the motility of Brugia malayi adults and microfilariae and on the fertility of B. malayi females was examined. Motility was reduced in adults after exposure to both drugs and worms were non-motile and dead within eight days. The motility of microfilariae was significantly reduced at all drug concentrations and ceased at concentrations of 2500 and 5000mug/mL. The motility of microfilariae released by females was reduced after exposure to both drugs, however ivermectin had a greater effect at concentrations between 170 and 5000mug/mL. Both drugs reduced the number of microfilariae released by females and within four days their release was inhibited. The presence of the bacterial endosymbiont Wolbachia was examined in adults and microfilariae after exposure to increasing concentrations of ivermectin and moxidectin. A decrease in wsp expression was correlated with increasing drug concentration.

Reduced genetic variation of an Onchocerca volvulus ABC transporter gene following treatment with ivermectin.

In some trichostrongyloid nematodes, the early stages of ivermectin (IVM) resistance have been characterized by a shift in allele frequency and reduced polymorphism at loci of P-glycoprotein genes, glutamate-gated chloride channel genes and gamma-aminobutyric acid receptor genes. Mass treatment with IVM is an integral component of the onchocerciasis control programmes. Genetic variation of an Onchocerca volvulus ABC transporter homologue (OvABC-3) from several populations in Africa was examined to determine whether an association exists between alleles of this gene and IVM treatment. Allelic variation in a non-treated population from Ghana showed this locus to be highly polymorphic. However, variability was reduced in IVM-treated populations. chi2 analysis of polymorph frequencies showed significant differences between untreated and treated samples collected in Ghana in 1999. There was less variability in this gene in samples collected in 2002 compared with the 1999 samples. In some treated populations, there appeared to be selection on OvABC-3-C. The observed reduction in variability could be expected in a control programme in which prevalence and intensity of infections are markedly reduced after years of vector control and IVM distribution. The reduction in polymorphism may not in itself indicate that these O. volvulus are IVM resistant, although it could indicate that selection for resistance is occurring.

Ivermectin imposes selection pressure on P-glycoprotein from Onchocerca volvulus: linkage disequilibrium and genotype diversity.

Widespread use of ivermectin (IVM) as part of the Onchocerciasis Control Program (OCP) in West Africa could influence the evolution of the human filarial parasite Onchocerca volvulus. Use of IVM, in some areas for 15 years, may have restricted genetic diversity of O. volvulus, resembling effects attributed to a population bottleneck. Large population-based chemotherapy programmes, such as the OCP, may impose strong selection pressure on parasites and an examination of possible genetic selection by IVM in O. volvulus is warranted. IVM is a substrate for P-glycoprotein; a homologue from O. volvulus (OvPGP) has been linked with IVM sensitivity. Linkage disequilibrium (LD) patterns of 28 genetic markers spanning the OvPGP locus were examined in 4 O. volvulus populations from the Volta Region of Ghana, West Africa. Reduced gene diversity, increased heterozygosity and an increase in the number of markers not in Hardy-Weinberg equilibrium were associated with increasing IVM treatment. The number of regions in LD decreased with treatment and with time. However, between 1999 and 2002, seven regions of OvPGP were always in complete LD, while surrounding areas showed a reduction in genetic variation. The use of IVM for onchocerciasis control has imposed strong selection on O. volvulus populations, reducing genetic variation and disrupting LD.

Identification of variant ABC-transporter genes among Onchocerca volvulus collected from ivermectin-treated and untreated patients in Ghana, West Africa.

Treatment with ivermectin (IVM) is known to cause a loss of polymorphism at certain loci of the beta-tubulin, gamma-aminobutyric-acid-receptor, glutamate-gated-chloride-channel and ATP-binding-cassette (ABC) transporter genes of IVM-resistant Haemonchus contortus. The genetic variation of four ABC-transporter homologues from Onchocerca volvulus was therefore investigated, to determine if any change in genetic polymorphism occurs in these genes following repeated treatment with IVM. Samples were collected in the Northern, Brong-Ahafo and Volta regions of Ghana, in 1999 and 2002; nodules containing adult O. volvulus were removed from subjects who had either received multiple IVM treatments or never taken IVM. The ATP-binding domains of four ABC-transporter genes (OvMDR-1, OvMDR-3, OvABC-1 and OvABC-6) were amplified from individual O. volvulus and examined for polymorphism, using single-strand-conformation-polymorphism (SSCP) analysis. In the samples collected in 1999, OvMDR-1 and OvABC-1 showed significant reduction in polymorphism following IVM treatment whereas OvABC-6 and OvMDR-3 were not found to be polymorphic. The samples collected in 2002 also showed a reduction in polymorphism for both OvMDR-1 and OvABC-1. Several single-nucleotide polymorphisms, which resulted in either amino-acid-replacement substitutions or nonsense mutations, were identified in the alleles of OvMDR-1 and OvABC-1.

In vitro nutritional requirements and metabolic products of pathogenic and nonpathogenic strains of Cryptobia salmositica: essential carbohydrates and amino acids.

Pathogenic and nonpathogenic strains of Cryptobia salmositica cultured in minimum essential medium (MEM) with several monosaccharides, disaccharides and amino acids were observed for differences in multiplication and motility. Metabolic end products (i.e. alanine, aspartate, carbon dioxide, lactate and pyruvate) were measured for logarithmically growing cells under aerobic conditions. The pathogenic strain of C. salmositica multiplied more readily in MEM supplemented with D(-)ribose, D(+)xylose, D(+)galactose, D(+)glucose, D(+)mannose and D(-)fructose. However, there were no significant differences in multiplication when the strains were cultured with the monosaccharide D(-)arabinose. The nonpathogenic strain multiplied significantly better than the pathogenic strain in the presence of the disaccharides alpha-lactose, maltose and sucrose. It also multiplied more readily when the amino acids L-glutamine and D(-)proline were added to MEM. The end products of carbohydrate catabolism under aerobic conditions were alanine, aspartate, carbon dioxide, lactate and pyruvate.

In vitro secretion of metabolic end-products by piscine haemoflagellates Cryptobia salmositica and C. bullocki (Kinetoplastida: Bodonidae) and the relationship of these products to the pH in the medium.

Pathogenic and nonpathogenic strains of Cryptobia salmositica Katz, 1951 and C. bullocki Strout, 1965 produced hydrogen peroxide, pyruvate and lactate under in vitro conditions in Minimum Essential Medium (MEM). As parasite number increased, the phenol red in the medium changed from red to yellow. This change was not associated with a decrease in pH, or an increase in pyruvate or lactate, but was correlated with an increased secretion of hydrogen peroxide. Parasites incubated at 10 degrees C in medium at pH 6.0, 6.5, 7.0 and 7.3 were active for about I week with decreasing activity in the absence of serum. Parasites in saline (pH 6.0, 6.5, 7.0 and 7.3) were nonmotile within 24 h and were dead in about 1 week. This suggests that these Cryptobia spp. are sensitive to changes in pH and require medium which is buffered, either with serum or Hepes.

The in vitro effects of isometamidium chloride (Samorin) on the piscine hemoflagellate Cryptobia salmositica (Kinetoplastida, Bodonina).

Isometamidium chloride (Samorin) is therapeutic in rainbow trout (Oncorhynchus mykiss) during preclinical and chronic cryptobiosis. However, the toxic mechanism of isometamidium on Cryptobia salmositica has not been elucidated. The objective of the present study was to examine the in vitro effects of isometamidium on C. salmositica. Under in vitro conditions, isometamidium chloride reduced the infectivity of C. salmositica suspended in whole fish blood. It accumulated rapidly in the kinetoplast (within 1 min) and caused disruption and decantenation of kinetoplast DNA. The in vitro cryptobiacidal activity of isometamidium was reduced when parasites were incubated in medium containing serum supplement, suggesting that isometamidium also binds to plasma proteins. Isometamidium altered glycoprotein receptors (epitopes) for antibodies on the surface of C. salmositica and thus protected some of the parasites from lysis by complement-fixing antibodies. In vitro oxygen consumption and carbon dioxide production decreased in drug-exposed C. salmositica, with increased products of glycolysis, i.e., lactate and pyruvate, after exposure to isometamidium. This suggests that some C. salmositica switched from aerobic respiration to glycolysis when the mitochondrion was damaged by isometamidium.

Therapeutic and prophylactic effects of isometamidium chloride (Samorin) against the hemoflagellate Cryptobia salmositica in chinook salmon (Oncorhynchus tshawytscha) and the effects of the drug on uninfected rainbow trout (O. mykiss).

A series of compounds (triphenylmethanes, thiazines, xanthenes, benzidines, phenanthridiniums, napthalamines, and diamidines) were screened for in vitro toxicity against Cryptobia salmositica. Isometamidium chloride (Samorin) was cryptobiacidal at low concentrations and was examined for therapeutic and prophylactic activities against C. salmositica in chinook salmon (Oncorhynchus tshawytscha). An intramuscular dose (1.0 mg/kg) of Samorin 3 weeks post-infection significantly reduced the parasitemia in adult chinook. A higher dose (2.5 mg/kg) eliminated the infection in 30% of adult fish and parasitemias were significantly reduced in the remaining infected fish. Juvenile chinook treated with 1.0 mg Samorin/kg at 2-3 weeks post-infection survived, while 100% of untreated control fish died from cryptobiosis. The high dose (2.5 mg/kg) was lethal to small fish (98.93 +/- 12.09 g) and 50% died within 24 h of treatment, while all large fish (168.38 +/- 13.87 g) survived. Samorin (1.0 mg/kg) did not affect growth, food consumption, complement, or hematocrit values in uninfected rainbow trout (O. mykiss).

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae).

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

The therapeutic use of isometamidium chloride against Cryptobia salmositica in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss injected intramuscularly with isometamidium chloride (0.01 or 0.1 mg kg-1) at 3 wk post-infection and given a booster 2 wk later had significantly lower parasitaemias than infected controls. Packed cell volume increased after treatment and remained higher than in infected controls. The concentration of isometamidium in plasma was highest at 2 wk after injection and then declined. An intramuscular dose of 1.0 mg kg-1 of isometamidium chloride at 1, 2 and 3 wk postinfection (preclinical) significantly reduced the parasitaemia in rainbow trout 2 wk after treatment. A booster at 9 wk postinfection (chronic disease phase) reduced the parasitaemia further in all fish. The packed cell volume in these fish was higher than in infected controls. Treatment at 5, 6, and 7 wk postinfection (acute disease) had no effects and parasitaemias in treated fish were higher than in infected controls; also, anti-Cryptobia salmositica antibodies and titres of complement-fixing antibody were higher in these than in infected controls. Incubation of immune plasma or complement with isometamidium for 3 h did not affect the lytic titres of complement-fixing antibodies nor rainbow trout complement.

The in vitro effects of crystal violet on the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida).

Crystal violet does not inhibit in vitro multiplication of a nonpathogenic strain of Cryptobia salmositica at low concentrations (0.01 microM and 0.001 microM) but multiplication is inhibited at higher concentrations (> or = 0.05 microM). In contrast, the pathogenic strain of C. salmositica does not multiply in vitro when incubated with crystal violet (0.001 microM, 0.01 microM and 0.05 microM). The infectivity of the pathogenic strain is significantly reduced after in vitro exposure to crystal violet. Crystal violet lyses C. salmositica (100.0 microM) and causes lesions on mitochondrial and nuclear membranes of the parasite. Pathogenic strains of Cryptobia salmositica and C. bullocki are more susceptible to lysis after in vitro exposure to crystal violet than are nonpathogenic strains of Cryptobia salmositica and C. catostomi.

Improved culture media for piscine hemoflagellates, Cryptobia and Trypanosoma (Kinetoplastida).

Increasing the Hepes buffer in minimum essential medium from 25 mM to 100 mM yielded a significantly larger number of Cryptobia salmositica. Cryptobia salmositica (pathogenic and nonpathogenic strains), Cryptobia bullocki, and Trypanosoma danilewskyi did not multiply either in heat-inactivated trout plasma (< or =25%) or in less than 10% fresh trout plasma. Both strains of C. salmositica multiplied better in 10% fresh trout plasma than in 25% heat-inactivated fetal bovine serum. In contrast, C. bullocki and T. danilewskyi multiplied better in 25% fetal bovine serum; 10% fetal bovine serum did not significantly reduce multiplication of C. bullocki. The nonpathogenic vaccine strain of C. salmositica cultured in 10% fresh trout plasma still protected rainbow trout from high parasitemia when challenged with the pathogen.

Protective antibodies and anamnestic response in Salvelinus fontinalis to Cryptobia salmositica and innate resistance of Salvelinus namaycush to the hemoflagellate.

Cryptobia-susceptible Salvelinus fontinalis vaccinated with a live Cryptobia salmositica vaccine were protected against C. salmositica and they were still protected 53 wk after their initial challenge. Parasites were lysed when they were incubated with immune plasma and complement and this confirms that complement-fixing antibody is an important part of the protective mechanism. Immunological memory was demonstrated in both vaccinated-challenged, and infected-recovered S. fontinalis as there were rapid and significant increases in complement-fixing antibody titers after parasite challenge. This indicates that S. fontinalis are capable of an anamnestic response. Most naive Salvelinus namaycush were not susceptible to C. salmositica infection, and their fresh plasma lysed C. salmositica under in vitro conditions. The lytic factor(s) was inactivated by heating the plasma prior to parasite incubation. This indicates that the alternate pathway of complement activation is likely the mechanism of innate resistance in S. namaycush.