Haemoparasite infection kinetics and the population structure of Theileria parva on a single farm in Uganda.

The development of sensitive PCR-based species-specific diagnostics and parasite genotyping methods offer the opportunity to provide important and detailed information on the infection dynamics of tick-borne disease. In this study we have exploited such tools to investigate the infection kinetics and parasite diversity within Theileria parva in a single farm in Uganda. Initial analysis of a sample of cattle showed high levels of infection with three Theileria species and Ehrlichia bovis, with most animals being infected with more than one pathogen. To study the infection dynamics, newborn calves were sampled longitudinally and it was shown that all animals became infected with T. parva, T. mutans, T. velifera and E. bovis with the average time to first infection being 53, 74, 116 and 109 days, respectively. However, the majority of these calves cleared the infections with T. parva and E. bovis but remained infected with the other two species of Theileria. In order to investigate the diversity of infecting genotypes of T. parva, samples from six calves were genotyped with a single mini-satellite marker at time points over a nine-month period. Each animal was infected with multiple different sets of genotypes and these were lost over different periods of time, implying that immunity is induced against particular infecting strains. To undertake a higher resolution analysis of parasite genotypes, samples from 30 calves were genotyped with a full panel of 12 micro- and mini-satellite markers but, due to the presence of mixed infections, only 16 samples could be used to generate parasite multi-locus genotypes (MLGs). A high degree of diversity of T. parva was seen on the farm, although some MLGs occurred more than once. Similarity analysis demonstrated a level of sub-structuring and the T. parva population was found to be in linkage disequilibrium. The basis for this high diversity coupled with apparent sub-structuring is discussed in relation to the possible causes.

Haemoparasite prevalence and Theileria parva strain diversity in Cape buffalo (Syncerus caffer) in Uganda.

Cape buffalo (Syncerus caffer) are considered to be an important reservoir for various tick-borne haemoparasites of veterinary importance. In this study we have compared the haemoparasite carrier prevalence in buffalo from four geographically isolated national parks in Uganda [Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP)]. Differences were seen in haemoparasite prevalence in buffalo from the four national parks. All the buffalo sampled in LMNP were carriers of Theileria parva however, buffalo from MFNP and KVNP, which are both located in the north of Uganda, were negative for T. parva. Interestingly, 95% of buffalo in the northern part of QENP were T. parva positive, however all buffalo sampled in the south of the park were negative. A high multiplicity of infection was recorded in all the buffalo found to be carrying T. parva, with evidence of at least nine parasite genotypes in some animals. Most of the buffalo sampled in all four national parks were carriers of T. mutans and T. velifera, however none were carriers of T. taurotragi, Babesia bovis, Babesia bigemina, Ehrlichia bovis or Ehrlichia ruminantium. All the buffalo sampled from LMNP were positive for T. buffeli and T. sp. (buffalo) however, buffalo from the parks in the north of the country (KVNP and MFNP) were negative for these haemoparasites. Anaplasma centrale and Anaplasma marginale were circulating in buffalo from all four national parks. T. parva gene pools from two geographically separated populations of buffalo in two of the national parks in Uganda (LMNP and QENP) were compared. The T. parva populations in the two national parks were distinct, indicating that there was limited gene flow between the populations. The results presented highlight the complexity of tick-borne pathogen infections in buffalo and the significant role that buffalo may play as reservoir hosts for veterinary haemoparasites that have the potential to cause severe disease in domestic cattle.

Detection of natural Trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of ITS1 rDNA.

Different species of trypanosomes may infect their mammalian hosts both singly or in combination. This study was undertaken to determine the trypanosome species that may be afflicting pigs in Uganda. Blood was collected from pigs of all ages and sexes from two districts, Kasese in Western and Jinja in Central Uganda. Of the 133 pig blood samples from Kasese that were tested for trypanosomes using the microhaematocrit centrifugation technique (MHCT), none was found to be infected. However, of the 253 pigs from Jinja district, nine were infected with trypanosomes of which three had T. vivax as determined by MHCT. However, application of the ITS1 rDNA PCR test revealed that eight pigs had T. vivax in mixed infections and one pig had T. vivax monolithic infection. These observations show that under certain circumstances, pigs may be important reservoirs for, as well as hosts to, T. vivax, contrary to earlier reports.

Characterisation of the Trypanosoma brucei rhodesiense isolates from Tanzania using serum resistance associated gene as molecular marker.

Serum resistance associated (SRA) gene has been found to confer resistance to the innate trypanolytic factor (TLF) found in normal human serum; thus allowing Trypanosoma brucei brucei to survive exposure to normal human serum. This study was carried out to examine the presence of SRA gene and identify the origin of T. b. rhodesiense isolates from three districts in Tanzania, namely Kibondo, Kasulu and Urambo. Twenty-six T. b. rhodesiense isolates and two references T. b. rhodesiense isolates from Kenya were examined for SRA gene using simple Polymerase Chain Reaction technique. The gene was found to be present in all 26 T. b. rhodesiense isolates including the two references isolates from Kenya. The SRA gene was confirmed to be specific to T. b. rhodesiense since it could not be amplified from all other Trypanozoon including T. b. gambiense; and gave an amplified fragment of the expected size (3.9kb), confirming that all these isolates were T. b. rhodesiense of the northern variant. Although the geographic distributions of T. b. gambiense and T. b. rhodesiense are clearly localized to west/central Africa and eastern Africa, respectively, natural movement of people and recent influx of large number of refugees into Tanzania from the Democratic Republic of Congo, could have brought T. b. gambiense in western Tanzania. The overlap in distribution of both of these pathogenic sub-species could result in erroneous diagnoses since both trypanosome sub-species are morphologically identical, and currently serologic methods have low specificity. Both the susceptible and resistant T. b. rhodesiense isolates possessed the SRA gene suggesting that there is no correlation between drug resistance and presence of SRA gene. The use of SRA gene helps to confirm the identity and diversity of some of the isolates resistant to various drugs.

Theileria parva live vaccination: parasite transmission, persistence and heterologous challenge in the field.

The 'Muguga cocktail' live vaccine, delivered by an infection and treatment protocol, has been widely deployed in Eastern, Central and Southern Africa to protect cattle against East Coast fever, caused by Theileria parva. The vaccine contains 3 component stocks (Muguga, Serengeti-transformed and Kiambu 5). In a previous study, parasites from vaccinated and unvaccinated animals were genotyped with a panel of micro- and minisatellite markers (Oura et al. 2004a) and it was shown that only the Kiambu 5 stock establishes a long-term carrier state but there was no evidence for the transmission of this stock. Also parasite genotypes different from the 3 component vaccine stocks were identified in vaccinated animals. We now report a follow-up study on the same farm, some 4 years after the initial vaccination, aimed at establishing the source of the novel parasite genotypes identified in vaccinated cattle, determining the longevity of the carrier state established by the Kiambu 5 vaccine stock and re-examining whether vaccine transmission can occur over a longer time-scale. To do this, samples were taken from vaccinated and unvaccinated cattle and the parasites were genotyped with a series of micro- and minisatellite markers. The data indicate that the vaccine stabilates contain at least 6 parasite genotypes, the Kiambu 5 stock can be detected in many but not all vaccinated cattle for up to 4 years and can be transmitted to unvaccinated cattle which share grazing and that some of the vaccinated animals become infected with local genotypes without causing overt disease.

Characterisation of the Trypanosoma brucei rhodesiense isolates from Tanzania using serum resistance associated gene as molecular marker

Serum resistance associated (SRA) gene has been found to confer resistance to the innate trypanolytic factor (TLF) found in normal human serum; thus allowing Trypanosoma brucei brucei to survive exposure to normal human serum. This study was carried out to examine the presence of SRA gene and identify the origin of T. b. rhodesiense isolates from three districts in Tanzania, namely Kibondo, Kasulu and Urambo. Twenty-six T. b. rhodesiense isolates and two references T. b. rhodesiense isolates from Kenya were examined for SRA gene using simple Polymerase Chain Reaction technique. The gene was found to be present in all 26 T. b. rhodesiense isolates including the two references isolates from Kenya. The SRA gene was confirmed to be specific to T. b. rhodesiense since it could not be amplified from all other Trypanozoon including T. b. gambiense; and gave an amplified fragment of the expected size (3.9kb), confirming that all these isolates were T. b. rhodesiense of the northern variant. Although the geographic distributions of T. b. gambiense and T. b. rhodesiense are clearly localized to west/central Africa and eastern Africa, respectively, natural movement of people and recent influx of large number of refugees into Tanzania from the Democratic Republic of Congo, could have brought T. b. gambiense in western Tanzania. The overlap in distribution of both of these pathogenic sub-species could result in erroneous diagnoses since both trypanosome sub-species are morphologically identical, and currently serologic methods have low specificity. Both the susceptible and resistant T.b. rhodesiense isolates possessed the SRA gene suggesting that there is no correlation between drug resistance and presence of SRA gene. The use of SRA gene helps to confirm the identity and diversity of some of the isolates resistant to various drugs.

Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Drug-resistance of Trypanosoma b. rhodesiense isolates from Tanzania.

OBJECTIVE: To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania. METHOD: We first screened 35 T. b. rhodesiense strains in the mouse model, for sensitivity to melarsoprol (1.8, 3.6 and 7.2 mg/kg), diminazene aceturate (3.5, 7 and 14 mg/kg), suramin (5, 10 and 20 mg/kg) and isometamidium (0.1, 1.0 and 2 mg/kg). A 13 isolates suspected to be resistant were selected for further testing in vitro and in vivo. From the in vitro testing, IC(50) values were determined by short-term viability assay, and MIC values were calculated by long-term viability assay. For in vivo testing, doses higher than those in the initial screening test were used. RESULTS: Two T. b rhodesiense stocks expressed resistance in vivo to melarsoprol at 5 mg/kg and at 10 mg/kg. These strains had high IC(50) and MIC values consistent with those of the melarsoprol-resistant reference strain. Another isolate relapsed after treatment with 5 mg/kg of melarsoprol although it did not appear resistant in vitro. One isolate was resistant to diminazene at 14 mg/kg and another was resistant at both 14 and 28 mg/kg of diminazene. These two isolates had high IC(50) values consistent with the diminazene-resistant reference strain. Two isolates relapsed at a dose of 5 mg/kg of suramin, although no isolate appeared resistant in the in vitro tests. Two isolates were resistant to isometamidium at 1.0 mg/kg and had higher IC(50) values. Two isolates were cross-resistant to melarsoprol and diminazene and one isolate was cross-resistant to suramin and isometamidium. CONCLUSION: The reduced susceptibility of T. b. rhodesiense isolates to these drugs strongly indicates that drug resistance may be emerging in north-western Tanzania.

Population genetic analysis and sub-structuring of Theileria parva in Uganda.

In recent years the population structures of many apicomplexan parasites including Plasmodium spp., Toxoplasma gondii and Cryptospordium parvum have been elucidated. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of another apicomplexan species (Theileria parva) to investigate the levels of diversity of this parasite and the role of genetic exchange in three geographically separate populations. The principal hindrance to carrying out such a study on field isolates was the high proportion of blood samples that contain multiple genotypes, making it impossible to determine the genotypes of the parasites directly. This problem was overcome by sampling only young indigenous calves between 3 and 9 months of age in which approximately 60% of the T. parva infected calves contained a single/predominant allele at each locus, making it possible to undertake population genetic analyses. Blood samples were collected from calves in three geographically distinct regions of Uganda and were analysed using 12 polymorphic mini and microsatellite markers that were evenly dispersed across the four chromosomes. We have identified 84 multilocus genotypes (MLG) from these samples, indicating high levels of diversity in the parasite. Analysis of linkage disequilibrium between pairs of loci provides evidence that the population in Lira district had an epidemic structure. The population in Mbarara was substructured containing two genetically distinct sub-groups and the larger sub-group also had an epidemic population structure. The population from Kayunga was in linkage disequilibrium. Genetic distances and Wrights fixation index (F(ST)) indicate that there is evidence for geographical sub-structuring between the Lira and the Kayunga populations.

The persistence of component Theileria parva stocks in cattle immunized with the 'Muguga cocktail' live vaccine against East Coast fever in Uganda.

The 'Muguga cocktail' live vaccine comprises three Theileria parva stocks (Muguga, Kiambu 5 and the buffalo-derived Serengeti-transformed) and has been used extensively in Eastern, Central and Southern Africa with an infection and treatment protocol to protect cattle against East Coast fever. We report the characterization of the three component vaccine stocks using a panel of polymorphic micro-satellite and mini-satellite markers and the development of a stock-derived PCR method that distinguishes two of the vaccine stocks. These markers, with the use of a recently developed Reverse Line Blot assay, have enabled us to address four important questions in relation to vaccination. First, how closely related are the vaccine stocks, secondly do all three stocks persist post-vaccination and induce a carrier state, thirdly is there evidence for the transmission of the vaccine stocks and fourthly does vaccination prevent infection with local genotypes? The results show that Muguga and Serengeti-transformed stocks are highly related but very distinct from Kiambu 5 that persists in vaccinated cattle establishing a carrier state. No evidence was obtained for the transmission of vaccine stocks to co-grazed animals, although these animals were infected with up to 8 different T. parva genotypes showing there was a significant level of tick challenge. Some of the vaccinated animals become infected with a subset of local genotypes providing evidence for limited vaccine 'breakthrough'.

Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda.

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1-2 x 10(3) parasites per ml of blood could be detected-a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.

A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva.

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.

Response of a T. b. rhodesiense stock with reduced drug susceptibility in vitro to treatment in mice and cattle.

In vivo drug susceptibility tests involving treatment of infected mice and cattle were performed on two trypanosome stocks, a T. brucei brucei and a T.b. rhodesiense, isolated in South Eastern Uganda. The T. b. rhodesiense stock had expressed reduced susceptibility to diminazene aceturate and isometamidium chloride in vitro, while the other, a T. b. brucei stock was susceptible. Diminazene aceturate at 14 mg/kg was not sufficient to cure all T. b. rhodesiense infected mice. Similarly, in the case of isometamidium chloride, 33% of infected mice treated with 2.0 mg/kg drug were not cured. In contrast, mice infected with the susceptible T. b. brucei and treated similarly with either drug were all cured. However, when cattle infected with the T. b. rhodesiense stock, or the susceptible T. b. brucei stock, or a 1:1 mixture of the two were treated with 7 mg/kg diminazene aceturate, they were all cured. Use of diagnostic PCR employing T. brucei specific primers confirmed that although the cattle had acquired infection pre-treatment, no trypanosome DNA amplification signal was demonstrated in the samples collected 60 days post-treatment. The reduced susceptibility of this T. b. rhodesiense, which could be demonstrated in mice as well as in culture, may indicate the existing potential for evolution of resistance in South Eastern Uganda.

Susceptibility of Ugandan Trypanosoma brucei rhodesiense isolated from man and animal reservoirs to diminazene, isometamidium and melarsoprol.

Thirty-six Trypanosoma brucei spp. stocks isolated from man and domestic animals in south-east Uganda were studied for susceptibility to diminazene, isometamidium and melarsoprol in vitro. All stocks were susceptible to melarsoprol. One T.b. rhodesiense stock isolated from a sleeping sickness patient showed reduced susceptibility to the veterinary drugs diminazene and isometamidium. More than 100 ng/ml diminazene or 0.78 ng/ml isometamidium were required to eliminate that stock during 10 days drug exposure. In contrast, the remaining stocks were eliminated by 0.8-6.3 ng/ml diminazene and 0.01-0.20 ng/ml isometamidium. Clones derived from the resistant T. b. rhodesiense stock showed reduced susceptibility to isometamidium and diminazene comparable to the parental population. Control of sleeping sickness by treatment of the animal reservoir could, therefore, face serious problems since drug-resistant stocks as reported here would most likely not be eliminated by recommended doses of diminazene and isometamidium.

Haemonchus contortus: the role of two beta-tubulin gene subfamilies in the resistance to benzimidazole anthelmintics.

The role of beta-tubulin genes in benzimidazole (BZ) resistance was investigated using one susceptible (S) and two resistant (Rt and Rc) strains of Haemonchus contortus. The Rt strain was isolated from the field on the basis of thiabendazole resistance. The Rc strain was derived from the S strain by treatment with cambendazole. cDNAs, derived from the S strain, encoding two isoforms of beta-tubulin (beta 12-16 and beta 8-9), alpha-tubulin and phosphofructokinase (Pfk) were used as probes for Southern hybridization analysis of genomic DNA digested by restriction enzymes. Genomic DNA was isolated from a pool of worms or single worms. The restriction-enzyme fragment length polymorphism (RFLP) differences among these strains depended on the enzyme and the probe used. When digested with Stu I or Hpa I, and probed under stringent conditions with beta 8-9 or beta 12-16, fewer fragments were seen in the Rt and Rc strains than in the S strain. Different hybridizing fragments were found in different individuals. The frequency of individuals bearing certain fragments hybridizing to beta 12-16 or beta 8-9 in the susceptible population was reduced significantly in the resistant populations. Some differences in RFLP between these strains were observed when probed with alpha-tubulin or Pfk, but the changes were not consistent with fragments being lost from the resistant strains as observed for beta-tubulin probes. These changes in RFLP pattern correlate with changes in the binding profiles of BZs and isoelectric isoform patterns reported previously for these strains. The data confirm that reduced heterogeneity within the population is associated with BZ resistance. Our results show that both the beta 8-9 and the beta 12-16 subfamilies of beta-tubulin are affected to a similar extent by this reduction in heterogeneity in a resistant population.

Expression of cloned beta-tubulin genes of Haemonchus contortus in Escherichia coli: interaction of recombinant beta-tubulin with native tubulin and mebendazole.

Two distinct beta-tubulin cDNA isotypes (beta 8-9 and beta 12-16) from Haemonchus contortus were expressed for the first time in Escherichia coli and characterised by their specific mebendazole (MBZ) binding and polymerization properties. Beta-tubulin was expressed without translational fusion to an E. coli sequence under the regulation of the tryptophan promoter in the pTrp2 vector. Beta-tubulin was produced in large amounts in insoluble 'inclusion bodies'. The inclusion bodies were purified and solubilised and the beta-tubulin renatured by treatment with urea followed by dilution with alkaline buffer and a shift to physiological pH. The yield was more than 10 mg of beta-tubulin per litre of cell culture. The recombinant tubulin produced was recognized in Western blot by specific anti-beta-tubulin antibodies. Tritiated MBZ binding to the recombinant H. contortus beta-tubulin was measured in the presence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [3H]MBZ high-affinity binding (HB) to 'pure' (no other eukaryotic protein present) beta 8-9 or beta 12-16 was observed. Enhanced high-affinity binding was observed when recombinant beta 8-9 or beta 12-16 were mixed and pre-incubated with whole supernatants or tubulin-enriched extracts from H. contortus. The enhancement was more than additive. Beta 12-16 bound more MBZ and caused a greater enhancement than beta 8-9. Mixing recombinant beta 8-9 or beta 12-16 with whole supernatants or tubulin-enriched fractions from H. contortus promoter polymerization at 37 degrees C. Use of 35S-labelled protein showed that the polymer contained recombinant tubulin. Western blot using specific anti-alpha-tubulin monoclonal antibodies showed that the polymer contained alpha-tubulin. Similarly the recombinant nematode beta-tubulin co-polymerized with tubulin from chicken brain. Our data suggest that the recombinant beta-tubulin can interact and copolymerize with parasite or chicken tubulin. Furthermore the interaction of recombinant nematode beta-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-binding sites. However, interaction of recombinant beta-tubulin with microtubule proteins from chicken brain did not result in the formation of high-affinity MBZ-binding sites.

Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy.

The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.

Beta-tubulin and benzimidazole resistance in the sheep nematode Haemonchus contortus.

We have compared benzimidazole (BZ) susceptible (s) and resistant (R) strains of Haemonchus contortus from sheep by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), Western blotting and ELISA techniques. The S strain bound more drug per mg protein than the R strain. BZ binding could be resolved into high-affinity and low-affinity binding. Low-affinity binding in parasite preparations devoid of tubulin was observed, but high-affinity binding occurred only in preparations containing tubulin. Resistance was associated with a decrease in the high affinity component. The S and R strains were shown by ELISA to contain similar total amounts of tubulin. By 2-D PAGE, the beta-tubulin isoform pattern of the S strain was different from that of the R strain, but the alpha-tubulin isoform patterns of the 2 strains were similar. BZ resistance was associated with a decrease in high-affinity BZ binding to tubulin and an alteration in beta-tubulin isoform pattern.

Specific interaction of benzimidazole anthelmintics with tubulin from developing stages of thiabendazole-susceptible and -resistant Haemonchus contortus.

The low- and high-affinity binding of tritiated benzimidazole anthelmintics (mebendazole and oxibendazole) to tubulin-containing supernatants derived from unembryonated eggs, third stage larvae or adult worms of thiabendazole-susceptible and -resistant strains of Haemonchus contortus were examined and compared. The displacement of these radioligands by unlabelled benzimidazoles (mebendazole, fenbendazole, thiabendazole and oxibendazole) also was examined. The binding affinity, K alpha, and maximum binding, Bmax, for the high-affinity binding were calculated by non-linear least-square iterative curve fitting using a computer programme (LIGAND) based on the exact mathematical model of ligand-receptor interactions. The K alpha was of the same order of magnitude (x 10(7) M-1) for the susceptible and resistant eggs, larvae and worms. Resistance was associated with a loss of high-affinity binding. There was a 2- to 5-fold loss of Bmax by the resistant strain. The eggs showed greater high-affinity binding per milligram of protein than the larvae which, in turn, showed greater high-affinity binding than the adult worms. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the tubulin content per milligram of protein decreased from egg, through larvae to adult worm. Cross-displacement studies indicated that different benzimidazole drugs interacted with the same receptor (tubulin) and that a rank order of affinity of the benzimidazole drugs could be inferred.

Specific interaction of benzimidazole anthelmintics with tubulin: high-affinity binding and benzimidazole resistance in Haemonchus contortus.

The specific (high-affinity) binding of tritiated benzimidazole [(3H]BZ) anthelmintics-mebendazole [(3H]MBZ) and oxibendazole [(3H]OBZ) to, and the specific displacement (inhibition) of these radioligands by unlabelled BZs (oxibendazole, mebendazole, oxfendazole, albendazole, fenbendazole and thiabendazole) from crude tubulin extracts prepared from thiabendazole (TBZ)-susceptible (S) or TBZ-resistant (R) strains of adult Haemonchus contortus, have been examined. The most striking difference between R and S was that the drug specifically bound at infinite ligand concentration (Bmax), was markedly reduced for the R strain, with no apparent change in association constant (Ka). Thus, resistance was associated with a loss of high-affinity receptors. TBZ-resistance was not associated with a change in low-affinity binding. There was a greater loss of high affinity receptors for [3H]OBZ than for [3H]MBZ. Using the displacement data. BZs were ranked according to their Ka and IC50 (concentration of BZ inhibiting 50% of radioligand binding) values. The Ka and IC50 values and the rank order of the BZs were approximately independent of the radioligand displaced or source (S or R) of the tubulin extracts used. The results are consistent with tubulin binding being the primary mechanism of action for all of these BZs.