Glen T. Coleman

Anthelmintic activity of cyclotides: in vitro studies with canine and human hookworms.

Hookworm infection is a leading cause of maternal and child morbidity in countries of the tropics and subtropics, as well as being an important parasite in companion-animal medicine. The cyclotides are a novel family of cyclic cystine knot containing peptides from plants that have been shown to possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes of sheep. In the current study we demonstrated the in vitro effects of three representative cyclotides, kalata B1, kalata B6 and cycloviolacin O14, on the viability of larval and adult life stages of the dog hookworm Ancylostoma caninum, and larvae of the human hookworm Necator americanus. The cyclotides showed significant anthelmintic activity towards both hookworm species. The different cyclotides showed similar patterns of relative activity as that seen previously with the livestock nematode species. This study demonstrates that cyclotides have promising activity in vitro against important parasites of companion animals and humans.

Acetylcholine receptor subunit genes from Ancylostoma caninum: Altered transcription patterns associated with pyrantel resistance ☆

The molecular mechanism of resistance to nicotinic agonist anthelmintics such as pyrantel and levamisole in nematodes of medical and veterinary significance is poorly understood. The identification of pyrantel-resistant isolates of the canine hookworm, Ancylostoma caninum, provides an opportunity to explore, at a molecular level, the mechanism of cholinergic resistance in a species that is a model for the human hookworms. Here we describe the cloning of three A. caninum genes orthologous to components of the pyrantel-sensitive nicotinic acetylcholine receptor in Caenorhabditis elegans (UNC-29, -38, -63). Analysis of mRNA levels by quantitative PCR was also performed on these genes, plus an additional three nicotinic acetylcholine receptor subunit genes thought not to be constituents of the pyrantel-sensitive receptor, for which a partial sequence was obtained. Gene sequences and mRNA levels were compared between two isolates of A. caninum showing either high- or low-level resistance to pyrantel (as shown previously by in vivo efficacy and in vitro comparative studies). While no polymorphisms of likely significance between the two A. caninum isolates were observed, quantitative analysis of transcription revealed significantly lower levels for the three putative pyrantel receptor subunits (AAR-29, -38 and -63) in the highly pyrantel-resistant isolate compared with the isolate with low-level resistance. In contrast, transcription of the three subunits thought not to constitute the pyrantel receptor (AAR-8, -15 and -19) was either not significantly different between the two isolates, or slightly higher in the highly-resistant isolate. This data suggests that reduced transcription of the mRNA coding for nicotinic acetylcholine receptor subunits that form the pyrantel-sensitive receptors may be a component of the pyrantel resistance mechanism in A. caninum.

Application of in vitro anthelmintic sensitivity assays to canine parasitology: Detecting resistance to pyrantel in Ancylostoma caninum

Resistance of the canine hookworm Ancylostoma caninum to anthelmintic therapy with pyrantel is an emerging problem in canine veterinary practice. Detecting anthelmintic resistance in parasites of pets is problematic because traditional resistance-monitoring techniques used with livestock parasites, such as the faecal egg count reduction test, are often impractical for use in small animals. We used two field-collected isolates of A. caninum in an abbreviated critical trial to test their pyrantel resistance status. The strains showed high-level and low-level resistance, with in vivo pyrantel efficacies of 28% and 71%, respectively. We noted a distinct worm density dependence effect on faecal egg count during the critical trial; egg counts in the dogs containing the low-level resistant isolate were 41% higher 6 days after drug treatment, despite the removal of 71% of the adult worms by the drug treatment. We then assessed four candidate in vitro assays for their ability to detect pyrantel resistance in A. caninum larvae, using these two isolates. The assays included a new format termed the larval arrested morphology assay (LAMA), based on observation of the effects of pyrantel on the body shape adopted by infective stage A. caninum larvae in vitro. Our data suggests that three of these assays, the LAMA, the larval motility assay (LMA), and larval feeding inhibition assay (LFIA) show promise with regards to detection of pyrantel resistance in A. caninum, but the complexity of the LFIA would likely limit its suitability for field studies. In vivo pyrantel efficacies of 28% and 71% in the two A. caninum isolates were associated with a 17-fold shift in the in vitro IC(50) values measured using the LAMA. Further testing with isolates of varying degrees of resistance is required to determine which of these assays is suitable as a rapid in vitro laboratory test for pyrantel resistance in A. caninum. The present study also indicates that potential exists for the novel LAMA or the LMA to be of use in detecting pyrantel resistance in the human hookworms, Necator americanus and Ancylostoma duodenale.

Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus.

While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite life cycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Large-scale monitoring of imidacloprid susceptibility in the cat flea, Ctenocephalides felis

Although on‐animal topical treatment with compounds such as imidacloprid has revolutionized the control of the cat flea, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae), the development of insecticide resistance is a continuing threat. As part of a highly co‐ordinated and unprecedented resistance monitoring programme for C. felis, 1437 flea isolates were collected by veterinary clinics in Australia, Germany, France, the U.K. and 29 states in the U.S.A. from 2002 to 2009. About 65% of the collections were made from June to October each year and 71% of the collections were from cats. Collections of flea eggs were sent to one of five different laboratories, where they were tested with a diagnostic dose of imidacloprid (3 p.p.m.) applied to larval flea‐rearing medium. Of the 1437 collections received, 1064 contained adequate numbers of eggs for testing. Of these isolates, untreated eggs failed to hatch in 22.7% and were not considered valid bioassays. Survival rates >5% and development of adult fleas (a threshold for further testing) occurred in only 22 isolates. They were re‐tested with the same diagnostic dose and none produced >5% adult emergence. Complete dose–response bioassays were performed on three of the isolates that had triggered a second test and produced slopes, intercepts and LC50 values similar to those for existing susceptible laboratory strains. Results confirmed sustained susceptibility of C. felis to imidacloprid, despite its widespread use for over a decade.

Canine vector-borne diseases in India: a review of the literature and identification of existing knowledge gaps

Despite the combination of favourable climate for parasites and vectors, and large populations of stray dogs, information concerning the epidemiology, diagnosis and management of canine vector-borne diseases in India is limited. However, with the countrys expanding economy and adaptation to western culture, higher expectations and demands are being placed on veterinary surgeons for improved knowledge of diseases and control. This review aims to provide an overview of the current state of knowledge of these diseases in India and identify existing knowledge gaps in the literature which need to be addressed. The available literature on this subject, although limited, suggests that a number of canine vector-borne diseases such as filariasis, babesiosis and ehrlichiosis are endemic throughout India, as diagnosed mostly by morphological methods. Detailed investigations of the epidemiology and zoonotic potential of these pathogens has been neglected. Further study is essential to develop a better understanding of the diversity of canine vector-borne diseases in India, and their significance for veterinary and public health.

HEMATOLOGY AND SERUM BIOCHEMISTRY OF THE BRUSH-TAILED ROCK-WALLABY (PETROGALE PENICILLATA)

In Australia the brush-tailed rock-wallaby (Petrogale penicillata) is the subject of a national recovery plan, and several sites have been selected for reintroductions. Condition of wild populations and individual animals can be monitored using hematologic and serum biochemistry analytes, and hematologic variables have been correlated with postrelease survival in other species. Prior to such monitoring, reference values for blood variables are required, but these data have not been available for the brush-tailed rock-wallaby. During four trapping periods from November 2004 to August 2005, 116 blood samples were collected from 44 brush-tailed rock-wallabies in a wild colony in southeast Queensland. Some variables varied with sex, age, method of restraint, lactation demands, and trapping period. After partitioning, when required, reference ranges for hematology and serum biochemistry variables were established. This study provides the most comprehensive serum biochemistry reference range for any macropodid marsupial yet published.

Comparative Pathology of Pulmonary Hydatid Cysts in Macropods and Sheep

The development and appearance of hydatid cysts of Echinococcus granulosus in experimentally infected tammar wallabies (Macropus eugenii) and sheep during the period 9-17 months post-infection (mpi) were studied. Cysts of unknown age were also examined from mature, naturally infected sheep. The cysts grew more rapidly and became fertile within a shorter period in wallabies compared with sheep. Cysts from the wallabies were larger in absolute size and were larger relative to the size of the lungs. Microscopical examination revealed that wallaby hydatid cysts developed in small bronchioles. Hydatid cysts in the wallabies had a thicker germinal membrane, with more nuclei and a thicker laminated layer (LL), than hydatid cysts of similar age found in sheep. In contrast, the adventitial layer was thicker in the ovine cysts, comprising a hyalinized layer of degenerate collagen and necrotic cellular debris surrounded by a layer of granulation tissue that was largely absent from lesions in the wallabies. Multilocular cysts were present in sheep, but not in wallabies. The greater thickness of the germinal membrane in wallaby cysts suggests greater parasite activity, which may explain the more rapid growth rate in this host, whereas the thicker adventitial layer in sheep cysts may be restrictive to growth while simultaneously protecting the hydatid from the host immune response. These differences in the parasite-host relationship between macropods and sheep may reflect the relatively recent introduction of the parasite into Australia.

Cystic echinococcosis in a wild population of the brush-tailed rock-wallaby ( Petrogale penicillata ), a threatened macropodid

Infection of small macropodids with the larval stage of Echinococcus granulosus can cause fatalities as well as significant pulmonary impairment and other adverse sequelae. The brush-tailed rock-wallaby (Petrogale penicillata) is a small macropodid listed as vulnerable on the IUCNs Red List of Threatened Species. This study used radiographic techniques to determine the prevalence and severity of pulmonary hydatid infection and growth rates of hydatid cysts in a wild population of this macropodid. The overall prevalence was 15.3% (9/59 animals) with 20.0% (8/40 animals) of adults infected. During the study period, the death of at least 1 infected animal was directly attributed to pulmonary hydatidosis. Rapid cyst growth occurred in some animals (up to 43% increase in cyst volume in 3 months). Cyst volume reduced lung capacity by up to 17%. Secondary pulmonary changes were uncommon but, in 1 animal, resulted in reduction in lung capacity by approximately 50%. Infection was associated with a higher blood urea concentration, but no significant differences in other blood variables were detected. These results indicate that hydatid infection may be a significant risk to threatened populations of small macropodids and should be addressed in conservation management plans for these animals.