Beta-tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations

The molecular mechanism of benzimidazole (BZ) resistance in cyathostomins of horses is still unclear. Previous studies revealed that the TTC or TAC polymorphism in codon 200 of the beta-tubulin isotype 1 gene is not as strictly correlated with BZ resistance as in trichostrongyles in sheep. To identify further sites of polymorphism within the beta-tubulin gene related to BZ resistance, complete complementary DNAs (cDNAs) encoding beta-tubulin of adult worms of Cylicocyclus nassatus, Cyathostomum pateratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicostephanus longibursatus, and Cylicostephanus goldi of a BZ-resistant cyathostomin population were characterized using specific primers. The cDNA sequence of each species spans 1,429 bp, encoding a protein of 448 amino acids. The interspecific identities are 95.2-99.6% at the nucleotide and 98.7-100.0% at the peptide level. The comparison of the amino acid sequences of individuals isolated from the BZ-resistant cyathostomin population with those from individuals of Cc. nassatus, Cy. coronatum, Cy. pateratum, and Cy. catinatum of a BZ-susceptible one showed differing amino acids in 11 positions. The commonness of a phenylalanine to tyrosine mutation at position 167 in all the 6 cyathostomin species isolated from a BZ-resistant population suggests its involvement in the molecular mechanism in BZ resistance.     

Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages

It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles.     

Evidence of p-glycoprotein sequence diversity in cyathostomins

P-glycoproteins (Pgps) are adenosine triphosphate-binding transporter proteins thought to be associated with multi-drug resistance in mammals and protozoans and have been suggested to be involved in the mechanism of ivermectin (IVM) resistance in Haemonchus contortus. Until now, resistance to IVM has not been reported in cyathostomins in horses in spite of its widespread and frequent use. Reasons for this might be differences in the molecular mechanism of the development of resistance. Based on this hypothesis, the present study was carried out to find homologues of Pgp in cyathostomins. A 416-bp polymerase chain reaction (PCR) product was generated using complementary DNA (cDNA) of Cylicocyclus elongatus and Cylicocyclus insigne and degenerate primers, located in the conserved Pgp nucleotide-binding domains. Resulting PCR products showed interspecific nucleotide and amino acid sequence identities of 73.3 and 76.8%, respectively. Specific primers were designed based on the Cc. elongatus sequence, and a PCR product of 268-bp was amplified from cDNA of single adults of Cylicocyclus radiatus, Cc. insigne, Cylicocyclus nassatus, Cc. elongatus, Cylicostephanus hybridus (2 individuals), Cylicostephanus goldi, Cyathostomum pateratum, Cyathostomum coronatum, and Cyathostomum catinatum. Two clusters of sequences were found representing 2 different internucleotide-binding domains (IBDs). A further distinct IBD is represented by the 416-bp PCR product of Cc. insigne. Therefore, a total of 3 clearly different sequences of the IBD were cloned and sequenced, suggesting that at least 2 Pgp genes exist in cyathostomins.     

Prevalence and abundance of equine strongyles (Nematoda: Strongyloidea) in tropical Australia

A postmortem survey of 57 horses in tropical northern Queensland revealed 41 (89%) infected with intestinal strongyles. Thirty-five strongyle species (8 large strongyles and 27 small strongyles [Cyathostominae]) were recorded of which 9 species are reported from Australia for the first time. The 14 most prevalent small strongyles were Cyathostomum catinatum (in 76% of horses), Cyathostomum coronatum (65%), Cyathostomum pateratum (33%), Cyathostomum labiatum (30%), Cylicostephanus calicatus (70%), Cylicostephanus longibursatus (67%), Cylicostephanus goldi (43%), Cylicostephanus minutus (26%), Cylicocylus nassatus (67%), Cylicocyclus leptostomus (41%), Cylicocylus insigne (41%), Cylicocyclus radiatus (33%), Cylicocyclus brevicapsulates (22%), and Poteriostomum imperidentum (24%). The remaining cyathostomes were each found in less than 15% of horses. The 4 most common large strongyles were Triodontophorus serratus (30%), Strongylus vulgaris (28%), Strongylus equinus, and Strongylus edentatus (both 22%). The number of species of small strongyles per horse showed a marked variation (mean 10.3, range 2-21) but bore no relationship to either the total number of strongyles per horse, age, sex, and breed of horse, or season. Total number of strongyles per horse (mean 15,890, range 20-165,000) was less than in recent surveys in Europe and the U.S.A. Most horses had low worm burdens, whereas a very small number were heavily infected. Ninety-seven per cent of the total strongyle counts were small strongyles. Strongylus species contributed just over 1%. Small numbers of large strongyles per horse were usual with T. serratus (mean 570), S. vulgaris (mean 330), and S. equinus (mean 330) the most numerous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parasitic helminths of the cecum and colon of equidae in Italy]

Intestinal helminths from coecum and colon were studied in 93 equidae including 40 horses, 36 donkeys and 17 mules. A total of 38 species, 36 nematodes and 2 cestodes, were identified as follows: 1) Triodontophorus serratus, 2) Triodontophorus brevicauda, 3) Strongylus equinus, 4) Strongylus edentatus, 5) Strongylus vulgaris, 6) Cyathostomum tetracanthum, 7) Cyathostomum coronatum, 8) Cyathostomum labiatum, 9) Cyathostomum labratum, 10) Cyathostomum alveatum, 11) Cyathostomum pateratum, 12) Cyathostomum catinatum, 13) Cyathostomum sagittatum, 14) Cylicodontophorus bicoronatus, 15) Cylicocyclus radiatus, 16) Cylicocyclus auriculatus, 17) Cylicocyclus elongatus, 18) Cylicocyclus nassatus, 19) Cylicocyclus insigne, 20) Cylicocyclus leptostomus, 21) Cylicostephanus calicatus, 22) Cylicostephanus poculatus, 23) Cylicostephanus minutus, 24) Cylicostephanus longibursatus, 25) Cylicostephanus hybridus, 26) Cylicostephanus goldi, 27) Cylicostephanus ornatus, 28) Cylicostephanus skrjabini, 29) Poteriostomum ratzii, 30) Gyalocephalus capitatus, 31) Parascaris equorum*, 32) Probstmayria vivipara, 33) Draschia megastoma*, 34) Habronema muscae*, 35) Habronema majus*, 36) Setaria equina*, 37) Anoplocephala perfoliata, 38) Paranoplocephala mamillana. The asterisked species are those not usually localized in the examined material. The most frequent parasites were found in all three hosts. Species 1, 4, 6, 9, 10, 12, 21, 22, 30 and 35 showed significant differences in prevalence between horses and donkeys, the mule generally having intermediate values. Multiple infections and total worm burden appear to decrease in older animals (> 15 years). Parasite associations occur mostly at random as expected from the values of the respective total prevalences. Some significant excesses on expected values were recorded but not significant deficits. The total worm burden increases with the number of parasite species and the increase appears to follow an exponential pattern.     

A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis

We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR-ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR-ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n=17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections.     

Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes

In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.     

Nucleotide sequences of three different isoforms of beta-tubulin cDNA from Chinese hamster ovary cells.

The complete cDNA sequences of two clones encoding beta-tubulin isotypes and the partial sequence of a third isoform from Chinese hamster ovary cells have been determined. The deduced amino acid sequences of the three isoforms show extensive homology to each other as well as with other alpha and beta-tubulin sequences from various species. These results provide evidence for the expression of three different isoforms of beta-tubulin in Chinese hamster ovary cells.     

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.     

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.