G. von Samson-Himmelstjerna

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.

Larvicidal and persistent efficacy of an imidacloprid and moxidectin topical formulation against endoparasites in cats and dogs.

Dogs and cats are often co-infected with endo- and ectoparasites. Roundworms of the genus Toxocara represent the most prevalent endoparasites in dogs and cats followed by hookworms of the genera Ancylostoma and Uncinaria. In the present study we have tested the anthelmintic efficacy of a new topical antiparasiticide which combines the insecticide imidacloprid and the macrocyclic lactone moxidectin for the simultaneous treatment and prevention of ecto- and endoparasitic infections in cats and dogs. Here, the efficacy against larval and immature stages of hook- and roundworms in cats was specifically investigated. Furthermore, the potential use of this product for the prevention of patent endoparasitic infections was addressed by demonstrating the persistent efficacy against Uncinaria stenocephala infections in dogs. Two controlled studies using experimentally infected cats showed 100% efficacy against third, fourth and immature adult stages of A. tubaeforme. Experimental infections with T. cati were eliminated to >97% and 91% concerning fourth and immature adult stages, respectively. Treatment of cats naturally infected with T. cati resulted in a complete removal of worm burdens. Evidence of persistent efficacy of the combination was obtained be treating dogs 18 days prior to infection with Uncinaria stenocephala larvae. In contrast to untreated control animals no worms were found in the intestines of the treated dogs 21 days post infection. No signs of local or systemic side effects were observed during these studies.

Flea allergy dermatitis in the cat: Establishment of a functional in vitro test

The purpose of this study was to develop a reliable and sensitive allergy test in the cat: The functional in vitro test (FIT) is monitoring exclusively those antibodies sensitizing basophiles and mastcells known as the prime initiators of type I allergies. By means of their Fc-receptors they accumulate antibodies of selected isotypes on their surface. Depending on their specificity these antibodies may bind the “fitting” antigens as bridging “allergens” causing the release of various mediators and, thus, the induction of type I allergy reactions. Histamine is one of these mediators and the only one being stored in considerable amounts in basophils and mastcells exclusively. The FIT was applied to blood specimens of a population of flea-infested laboratory cats. Individual blood samples were washed and divided into subunits. Treatments comprising spontaneous, physical, antibody and antigen mediated histamine release were applied. For antigen induced release the flea antigen was titrated at final concentrations between 1 and 1 * 10-6 μg/ml. The amount of released histamine was quantified by a radio immuno assay (RIA). Blood samples from individual cats showed interdividual variations of histamine contents as observed in humans and mice. The allergen preparation of Ctenocephalides spp. proved to be free of unspecific triggering of feline basophils. In the concentrations tested it caused dose dependent and individually very different reactions. Thus, this functional in vitro test system might provide a sensitive and reliable monitoring for type I allergies in the cat. It still requires further investigations regarding evaluation of allergen released histamine in order to obtain clinically relevant results.

EVIDENCE OF P-GLYCOPROTEIN SEQUENCE DIVERSITY IN CYATHOSTOMINS

P-glycoproteins (Pgps) are adenosine triphosphate–binding transporter proteins thought to be associated with multidrug 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.

Putative G protein-coupled receptors in parasitic nematodes - potential targets for the new anthelmintic class cyclooctadepsipeptides?

Parasitic nematodes cause major problems in livestock animals. Resistances to the most commonly used drugs are arising. The cyclooctadepsipeptide emodepside belongs to a new class of anthelmintics. A receptor for emodepside, Hc110–R, was previously identified in Haemonchus contortus. We have identified the complete coding sequences of putative orthologues in Cooperia oncophora and Ostertagia ostertagi, tri–chostrongyles in cattle. The putative receptors were named depsiphilins. The deduced amino acid sequence of C. oncophora depsiphilin has a similarity of 91% to the O. ostertagi sequence. The similarity of both the C. oncophora and O. ostertagi depsiphilin to Hc110–R is 89%, based on the amino acid sequence. The depsiphilins share 46% identity with the latrophilin–like protein 1 in Caenorhabditis elegans and 47% identity with a hypothetical protein in Caenorhabditis briggsae. Hc110–R and the latrophilin–like proteins of C. elegans were previously reported to be putative G protein–coupled receptors (GPCRs) and to be related to mammalian latrophilins. A seven transmembrane domain, a GPCR proteolytic site, and other conserved domains characteristic of receptors of the latrophilin group were identified within the depsiphilins. Therefore it seems reasonable to allocate the depsiphilins to the previously described latrophilins and latrophilin–like proteins.

Estudio sobre resistencia frente a los bencimidazoles de pequeños estróngilos (Cyathostominae) del equino en el sur de Chile

La presencia de resistencia de los pequenos estrongilos a los benzimidazoles se determino, en un total de 100 equinos, en tres planteles del Sur de Chile mediante las pruebas de reduccion de oviposicion (faecal egg count reduction test, FECRT) y de eclosion de huevos (egg hatch assay, EHA). A cada equino se le tomaron muestras fecales desde el recto siete dias antes y siete dias despues de un tratamiento con fenbendazol (Panacur[marca registrada]) con el objeto de determinar un posible aumento del grado de resistencia. Las medias aritmeticas de los recuentos de huevos se distribuyeron entre 476.7 (±356.7) y 1095.3 (±755) hpg antes, y entre 137.1 (±171.8) y 725 (±481.3) hpg despues del tratamiento. Mediante la prueba FECR se determino resistencia en los tres planteles al encontrar reducciones de 27% (±33), 26.5% (±26.9) y 83.9% (±22.8). Por medio de la prueba EHA se constato resistencia antes del tratamiento en un plantel (DL[subindice 50] = 0.141 TBZ/ml) y, despues del tratamiento, en dos planteles (DL [subindice 50] = 0.149 y 0.158 [mi griega]g TBZ/ml respectivamente).

Improved in vitro cultivation of larvae of the bovine lungworm Dictyocaulus viviparus

Abstract Parasitic larvae of Dictyocaulus viviparus are of major importance for the development of immunity in cattle. The conditions for in vitro cultivation of D. viviparus larvae as well as their morphology have thus far been only poorly investigated. Exsheathed larvae were cultivated in vitro in RPMI-1640 (Gibco-BRL, pH 7.2) containing 20% newborn Calf serum, 200 U Moronal/ml, and 200 U penicillin/streptomycin/ml. Incubation was performed at 39.5 °C at 0, 5%, 10%, and 20% CO2. Average development rates to third-moult (3 M) or fourth-stage (L4) larvae at 5% CO2 incubation were 8.33% (SD ± 7.76%), 22.52% (SD ± 13.09%) at 10% CO2, and 38.01% (SD ± 15.63%) at 20% CO2. These differences were statistically significant. Some morphological features of these larvae are described.

Monitoring of basophil sensitization to antigens of the cat flea (Ctenocephalides felis felis): a new tool for the diagnosis of feline flea bite hypersensitivity?

Suitability of blood basophils for in vitro diagnosis of flea allergy dermatitis (FAD) or flea bite hypersensitivity was studied in cats. A functional in vitro test (FIT) for sensitized type I allergic effector cells was used to evaluate the degree and kinetics of in vivo basophil sensitization against flea antigens in cats under long-term flea exposure. FIT results were compared with intradermal (IDT) and serological testing. Before, during, and after weekly repeated exposure to Ctenocephalides felis; 14 cats were repetitively FIT-assessed for general and flea-specific sensitization. In three cats, flea-specific sensitization was seen before and throughout flea exposure. Five cats, although generally sensitized, never developed a flea-specific sensitization. Six cats initially FIT-negative became sensitized for flea antigen during flea infestation. Induction, upregulation, and binding of C. felis-specific sensitizing antibodies to basophils during flea challenge may explain the developing sensitization in these cats. Strong discrepancies between the levels of flea-specific circulating IgE and basophil sensitization contrasted comparable results for basophil and mast cell sensitization using FIT and IDT, respectively. Hence, the FIT might provide an immunological supplement to the clinical diagnosis of FAD in cats by elucidating the state of basophil-sensitization to flea antigens. And it may be a comfortable alternative to IDT.

Polymerase chain reaction-based construction of cDNA libraries from minute amounts of third-stage and fourth and fifth-stage larvae of Dictyocaulus viviparus

Abstract A strategy is described for the amplification and cloning of cDNA from minute amounts of Dictyocaulus viviparus larvae. Initially, third-stage larvae (L3) were used to establish the procedure. Amplification of cDNA synthesized from approximately 400 ng total RNA from 5,000 L3 generated products that were more than 800 bp in length. The unidirectional cloning of amplified cDNA products led to the construction of a UNI ZAP cDNA library with 1×106 clones. Screening with a homologous oligo(dT)-primed digoxigenin-labeled cDNA probe as well as sequencing of seven randomly picked clones confirmed the successful cloning of lungworm cDNA. Subsequently, approximately 600 ng total RNA was isolated and polymerase chain reaction (PCR) products of up to 2,400 bp were amplitied from 400 fourth- and fifth-stage larvae (L4/L5). Cloning of these products resulted in a L4/L5 cDNA library of D. viviparus consisting of 5×105 recombinant clones. In all, 11 clones were randomly picked and sequenced, all revealing typical mRNA/cDNA characteristics. Comparison of the predicted amino acid sequence of the 5′ end of clone DvL5/7 revealed 100% homology with the actin gene of several other helminths.

Analysis of codon usage in β-tubulin sequences of helminths

Codon usage bias has been shown to be correlated with gene expression levels in many organisms, including the nematode Caenorhabditis elegans. Here, the codon usage (cu) characteristics for a set of currently available β-tubulin coding sequences of helminths were assessed by calculating several indices, including the effective codon number (Nc), the intrinsic codon deviation index (ICDI), the P2 value and the mutational response index (MRI). The P2 value gives a measure of translational pressure, which has been shown to be correlated to high gene expression levels in some organisms, but it has not yet been analysed in that respect in helminths. For all but two of the C. elegans β-tubulin coding sequences investigated, the P2 value was the only index that indicated the presence of codon usage bias. Therefore, we propose that in general the helminth β-tubulin sequences investigated here are not expressed at high levels.Furthermore, we calculated the correlation coefficients for the cu patterns of the helminth β-tubulin sequences compared with those of highly expressed genes in organisms such as Escherichia coli and C. elegans. It was found that β-tubulin cu patterns for all sequences of members of the Strongylida were significantly correlated to those for highly expressed C. elegans genes. This approach provides a new measure for comparing the adaptation of cu of a particular coding sequence with that of highly expressed genes in possible expression systems.Finally, using the cu patterns of the sequences studied, a phylogenetic tree was constructed. The topology of this tree was very much in concordance with that of a phylogeny based on small subunit ribosomal DNA sequence alignments.