Nicholas Sangster

Geometric means provide a biased efficacy result when conducting a faecal egg count reduction test (FECRT)

The process of conducting a faecal egg count reduction test was simulated to examine whether arithmetic or geometric means offer the best estimate of efficacy in a situation where the true efficacy is known. Two components of sample variation were simulated: selecting hosts from the general population which was modelled by the negative binomial distribution (NBD), and taking an aliquot of faeces from the selected host to estimate the worm egg count by assuming a Poisson distribution of sample counts. Geometric mean counts were determined by adding a constant (C) to each count prior to log transformation, C was set at 25, 12 or 1. Ten thousand Monte Carlo simulations were run to estimate mean efficacy, the 2.5% (lower) and the 97.5% (upper) percentile based on arithmetic or geometric means. Arithmetic means best estimated efficacy for all different levels of worm aggregation. For moderate levels of aggregation and with C=1 the geometric mean substantially overestimated efficacy. The bias was reduced if C was increased to 25 but the results were no better than those based on arithmetic means. For very high levels of aggregation (over-dispersed populations) the geometric mean underestimated efficacy regardless of the size of C. It is recommended that the guidelines on anthelmintic resistance be revised to advocate the use of arithmetic means to estimate efficacy.

Confirmation of Fasciola hepatica resistant to triclabendazole in naturally infected Australian beef and dairy cattle

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Haemonchus contortus infection in sheep: Parasite fecundity correlates with worm size and host lymphocyte counts

Two experiments were conducted to elucidate the timing and nature of the sheep immune response to Haemonchus contortus (Barbers pole worm). The first experiment examined the establishment of H. contortus populations and the immune response by comparing a bolus infection of third-stage larvae in naïve sheep with a group previously primed by a trickle infection. The second experiment used staggered doses of ivermectin-resistant larvae to compare the development of adult worms during different durations of trickle infection with ivermectin-sensitive larvae. Infections successfully generated pathological signs of haemonchosis such as anaemia. Image analysis software was used to measure the area and perimeter of worms collected at post-mortem, and the number of eggs present in individual adult females (fecundity) was significantly correlated with worm size. A significant inverse correlation was found between blood lymphocyte counts and worm fecundity. The absence of correlation between worm fecundity and other leukocyte and erythrocyte counts highlighted the specificity of the lymphocyte response. This is the first report of a link between haematology profiles and worm fecundity in haemonchosis. The correlation observed between adult worm size and egg content leads to the hypothesis that egg production in H. contortus is limited by immune regulation of worm size and presumably growth. Mean worm size and fecundity declined as sheep received more prolonged trickle infections before necropsy, confirming previous reports that immune responses to adult worms are enhanced by ongoing larval challenge. Immunohistochemical results showed trends consistent with a Th2 (humoral) immune response which has been implicated in reducing nematode burdens in several species.

Comparative kinetics of serological and coproantigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole

Three methods of diagnosing Fasciola hepatica (F. hepatica) infection (a coproantigen ELISA, Bio-X Diagnostics, Belgium, Faecal Egg Count (FEC), and a serum IgG ELISA,Bio-X Diagnostics, Belgium) were evaluated in artificially infected cattle, with and without drug treatment. Specifically, the potential value of the coproantigen ELISA in the quantitation of F. hepatica infection was sought. Twelve steers were each infected with 100, 200 or 500 metacercariae (n=4 cattle/group). On day 84, post infection (PI), 2 animals from each group were treated orally with triclabendazole (TCBZ). Faecal and blood samples were collected weekly after infection from all animals, as well as over 5 consecutive days (days 105-109 PI) for the six animals remaining infected to determine the repeatability of these assays. Cattle were killed 126 days PI and the coproantigen, FEC and IgG levels were compared with the number of fluke recovered. Animals first tested positive for infection with the serum ELISA, with 11/12 animals positive on day 28, and IgG responses increased to day 42 PI. The coproantigen ELISA was first positive on day 42 (3/12 animals), with all animals positive by day 56 PI. The first F. hepatica egg was detected on day 49 from an animal infected with 500 metacercariae; however only on one occasion (day 84) did all animals return positive FEC. Within one week of treatment with TCBZ, all six treated animals had returned to negative status by coproantigen ELISA and FEC whereas IgG levels persisted. Weekly variation in both coproantigen level and FEC was evident throughout the trial. Results from the consecutive daily collections varied greatly between days for both methods, with 2-6-fold differences in coproantigen levels and 2-4-fold variation in FEC. Strong correlations were observed between fluke burdens (day 126) and day 125 coproantigen levels (R(2)=0.8718) and FEC (R(2)=0.8368). The coproantigen ELISA was more sensitive than FEC (FEC displayed false negatives) and detected infection earlier. This ELISA showed good correlation to fluke burdens in these cattle and has promise as a test for detecting low fluke burdens.

Genomic analyses of Haemonchus contortus infection in sheep : Abomasal fistulation and two Haemonchus strains do not substantially confound host gene expression in microarrays

To determine whether fistulation and differing strains of Haemonchus contortus complicate genome analysis of the host response to infection, two pilot experiments examined parasite development and gene expression in the abomasal mucosa of parasitised sheep. No significant differentially-expressed genes were detected in a comparison between ivermectin-susceptible McMaster and ivermectin-resistant CAVR strains of H. contortus. This demonstrated that the sheep response was not significantly altered by the ivermectin-resistance status of the parasite. However, sheep infected with McMaster strain had a significantly lower proportion of larvae and a higher mean FEC at post-mortem than sheep infected with CAVR, suggesting that McMaster larvae advance to patency faster than CAVR larvae. Abomasal fistulation resulted in significant upregulation of three genes and significant downregulation of two genes. Fistulated sheep had significantly lower FEC than the other groups but the proportion of larvae at post-mortem was not significantly different to other groups infected with the same strain (CAVR). Hence fistulation does not alter establishment of the CAVR isolate, but may slow its progression to patency. The observation that different H. contortus strains and abomasal fistulation induced minimal changes in mucosal gene expression validated the design of a subsequent experiment (manuscript in preparation) where sequential biopsies taken during infection were analysed by microarray to describe the molecular responses which inhibit larval establishment.

Sequential microarray to identify timing of molecular responses to Haemonchus contortus infection in sheep.

Anthelmintics are currently the most common method of worm control. The emergence of worms with multiple-drug resistance and issues of residues in the food chain make alternative parasite control measures a priority. To develop improved and sustainable methods for controlling Haemonchus contortus such as genetic selection of resistant sheep, a better understanding of the host-parasite relationship is required. A trial was undertaken using sheep surgically implanted with abomasal fistulas to enable sequential biopsy of the abomasal mucosa during trickle infection with two strains of H. contortus. These were ivermectin-resistant CAVR and ivermectin-sensitive McMaster. From a gross parasitology perspective, this approach enabled the effect of developing immunity to be observed on both the establishment and maturation of two CAVR doses within and between groups. Since the only difference in parasite treatment between the groups was the staggering of the two CAVR doses, microarray results from biopsies taken on the same day in different groups were combined and compared between different biopsy dates to observe differential gene transcription over time. Differential gene transcription was detected by comparing transcription in our array data between different biopsy dates using a low P value screen (P<0.01) and by compiling a list of 82 immunoparasitology-related genes and examining transcription in this list with a higher P value screen (P<0.05). Our microarray data were validated in silico by comparison with intelectin 2, trefoil factor 3, calcium activated chloride channel and mucin 5 from other gene transcription studies and with phenotypic data such as the response by gammadelta T cells and immunoglobulins to H. contortus. The first four genes are involved in non-specific responses to infection and mucosal healing. These were upregulated at the early time points and intelectin 2 remained prominent throughout the trial. As the trial progressed, immunoglobulin genes became strongly upregulated. These included IgCgamma IgG2a heavy chain constant region, IGHE immunoglobulin heavy constant epsilon and IGHM immunoglobulin heavy constant mu.

The in vitro assay profile of macrocyclic lactone resistance in three species of sheep trichostrongyloids

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The efficacy of ivermectin, pyrantel and fenbendazole against Parascaris equorum infection in foals on farms in Australia

This study was performed to estimate the prevalence of patent Parascaris equorum infections and determine the efficacy of ivermectin, pyrantel and fenbendazole against P. equorum infection in foals on farms in southern Australia. Foals aged >3 months on five farms in the south-western slopes region of New South Wales were used. Faeces were collected from each foal and foals with a P. equorum faecal egg count (FEC) of >100 eggs per gram (EPG) were used to measure anthelmintic efficacy using the FEC reduction (FECR) test, after random allocation to a control group or an ivermectin, pyrantel embonate or fenbendazole treatment group. Treatment was administered on day 0 and faeces were collected on day 14 and a FEC was performed. For determination of anthelmintic efficacy, FECRs and lower 95% confidence intervals (LCL) were calculated using previously described methods, based on individual or group FECRs. P. equorum populations were considered susceptible when FECR was >90% and LCL >90%, suspected resistant when FECR was FECR was 80-90% and LCL <90% and resistant when FECR was <80% and LCL <90%. A Poisson distribution quality control method was applied to the data to remove suspected erroneous FECR results. Prevalence of patent P. equorum infection was 58.3% (147/252 foals) and 89 foals on 5 farms were included in the FECR study. Resistance of P. equorum to ≥ 1 anthelmintic was present on all five farms prior to and on four farms after application of the quality control method. Two farms had evidence of multiple drug resistance. Ivermectin was effective and ineffective on two and three farms, respectively. Fenbendazole was effective on two farms, equivocal on one farm and ineffective on one farm. Pyrantel embonate was effective on three farms and ineffective on one farm. These data indicate that anthelmintic-resistant P. equorum populations are present on farms in Australia and multiple drug resistance may occur on individual farms.

Evidence for high genetic diversity of NAD1 and COX1 mitochondrial haplotypes among triclabendazole resistant and susceptible populations and field isolates of Fasciola hepatica (liver fluke) in Australia.

In recent years, the global incidence of Fasciola hepatica (liver fluke) infections exhibiting resistance to triclabendazole (TCBZ) has increased, resulting in increased economic losses for livestock producers and threatening future control. The development of TCBZ resistance and the worldwide discovery of F. hepatica population diversity has emphasized the need to further understand the genetic structure of drug susceptible and resistant Fasciola populations within Australia. In this study, the genetic diversity of liver flukes was estimated by sequencing mitochondrial DNA (mtDNA) encoding the NAD1 (530 bp) and COX1 (420 bp) genes of 208 liver flukes (F. hepatica) collected from three populations: field isolates obtained from abattoirs from New South Wales (NSW) and Victoria (Vic); three TCBZ-resistant fluke populations from NSW and Victoria; and the well-established TCBZ-susceptible Sunny Corner laboratory isolate. Overall nucleotide diversity for all flukes analysed of 0.00516 and 0.00336 was estimated for the NAD1 and COX1 genes respectively. Eighteen distinct haplotypes were established for the NAD1 gene and six haplotypes for the COX1 gene, resulting in haplotype diversity levels of 0.832 and 0.482, respectively. One field isolate showed a similar low level of haplotype diversity as seen in the Sunny Corner laboratory isolate. Analysis of TCBZ-resistant infrapopulations from 3 individual cattle grazing one property revealed considerable sequence parasite diversity between cattle. Analysis of parasite TCBZ-resistant infrapopulations from sheep and cattle revealed haplotypes unique to each host, but no significant difference between parasite populations. Fst analysis of fluke populations revealed little differentiation between the resistant and field populations. This study has revealed a high level of diversity in field and drug resistant flukes in South-Eastern Australia.

Haemonchus contortus: Development of a two-step, differential-display PCR to detect differential gene expression in nematodes from immune and naive sheep

Haemonchus contortus is a blood-feeding nematode which parasitizes the abomasum of sheep and represents a serious constraint to sheep production. Anthelmintics are currently the most common method of worm control but the worldwide development of multiple-drug resistance and issues of residues in the food chain make alternatives to anthelmintics a priority. Biotechnology-driven solutions to parasitism include vaccines and silencing of genes regulating nematode development. To pursue gene targets that may be suitable for parasite control, a two stage differential-display PCR (dd-PCR) approach was developed to observe differential gene expression between Haemonchus from immune and control sheep. Twenty-four reproducible differentially-expressed bands were identified in 60 pairs of dd-PCR comparisons. The first of these cloned and sequenced corresponded to the H. contortus 60S ribosomal protein L35A. The remaining bands are being cloned and validated and may provide new targets for parasite control.