Feasibility of Inducing Overlap Immunologic Competence in Gallinaceous Birds with Ascardia dissimilis and A. galli

Chickens and turkeys are routinely infected with the roundworms Ascaridia galli and A. dissimilis, respectively. The current study was conducted to gather basic information on these worms and to determine whether heterologous infections (chicken worms in turkeys and turkey worms in chickens) would be successful. Chickens and turkeys were obtained at day of hatch, brooded to 7 days of age, and placed in pens (25/pen) according to infection as received at 7 days of age: homologous, heterologous and control (no infection). Bird weights, mortalities, and feed efficiencies were monitored for 3 weeks postinfection, at which time all birds were killed for parasite collection and counting. Feed efficiency, a parameter more adequately measured in large-scale studies, did not vary between experimental groups. Bird weights at necropsy varied significantly (P \u3c 0.05) between groups only for the turkeys, with homologous infection (A. dissimilis) birds weighing less than controls. All induced, homologous, and heterologous infections were successful. Rates of establishment, however, were significantly (P \u3c 0.05) depressed for each heterologous model. Total A. dissimilis numbers were only 55% as great as those for A. galli in chickens [geometric means (GMs) of 13.2 versus 24.2], and total A. galli numbers were only 56% as great as A. dissimilis numbers in turkeys (GMs of 5.6 versus 10.0). Given the fact that heterologous infections were successful, albeit reduced, in both types of birds (infections that included tissue-phase forms), additional studies are planned to determine whether these infections might induce interspecies (overlapping) immune competence in the host and aid in reducing natural parasitisms to levels with no economic impact

Contrasting patterns of isotype-1 β-tubulin allelic diversity in Haemonchus contortus and Haemonchus placei in the southern USA are consistent with a model of localised emergence of benzimidazole resistance

The benzimidazoles are one of the most important broad-spectrum anthelmintic drug classes for parasitic nematode control in domestic animals and humans. They have been widely used in livestock, particularly in small ruminants for over 40 years. This has resulted in widespread resistance in small ruminant gastrointestinal nematode parasite species, especially Haemonchus contortus. Benzimidazole resistance mutations have also been reported in Haemonchus placei, but only at low frequencies, suggesting resistance is at a much earlier stage of emergence than is the case for H. contortus. Here, we investigate the haplotype diversity of isotype-1 β-tubulin benzimidazole resistance mutations and the population genetic structure of H. contortus and H. placei populations from sheep and cattle from the southern USA. Microsatellite genotyping revealed a low level of genetic differentiation in six H.placei and seven H. contortus populations examined. This is consistent with several previous studies from other regions, mainly in H. contortus, supporting a model of high gene flow between parasite populations. There was a single F200Y(TAC) haplotype present in all six H. placei populations across Georgia, Florida and Arkansas. In contrast, there were at least two different F200Y(TAC) haplotypes (up to four) and two different F167Y(TAC) haplotypes across the seven H. contortus populations studied. These results provide further evidence to support a model for benzimidazole resistance in Haemonchus spp, in which resistance mutations arise from a single, or the small number of locations, in a region during the early phases of emergence, and subsequently spread due to animal movement

The presence of benzimidazole resistance mutations in Haemonchus placei from US cattle

Haemonchus populations were collected from cattle from mid-western and eastern Southern US (four and six populations, respectively) to determine the relative prevalence of Haemonchus contortus and Haemonchus placei and the frequency of the three isotype-1 β-tubulin polymorphisms associated with benzimidazole resistance. A minimum of 32 individual adult worms were genotyped at position 24 of the rDNA ITS-2 for each population to determine species identity (296 worms in total). One population from Georgia was identified as 100% H. contortus with the remaining nine populations identified as 100% H. placei. For the H. contortus population, 29 out of 32 worms carried the P200Y (TAC) isotype-1 β-tubulin and 2 out of 32 worms carried the P167Y (TAC) benzimidazole resistance associated polymorphisms respectively. For H. placei, six out of the nine populations contained the P200Y (TAC) isotype-1 β-tubulin benzimidazole resistance associated polymorphism at low frequency (between 1.6% and 9.4%) with no resistance associated polymorphisms being identified at the P198 and P167 codons. This is the first report of the P200Y (TAC) isotype-1 β-tubulin benzimidazole resistance associated polymorphism in H. placei. The presence of this mutation in multiple independent H. placei populations indicates the risk of resistance emerging in this parasite should benzimidazoles be intensively used for parasite control in US cattle

Reflections and future directions for continued development and refinement of guidelines for anthelmintic efficacy studies

This reflection paper complements the WAAVP (World Association for the Advancement of Veterinary Parasitology) general anthelmintic efficacy guideline, which outlines the general principles of anthelmintic efficacy evaluation across all animal host species. It provides background to the recommendations made in the WAAVP general anthelmintic efficacy guideline, with insights into the discussions leading to specific recommendations in the general guideline or the absence thereof. Furthermore, this paper discusses recent technological advancements with potential value to the evaluation of anthelmintic efficacy that may be considered for future versions of the general or species-specific guidelines if supported by sufficient levels of evidence. Finally, it also identifies potential research questions, such as the statistical approach for comparing worm counts between groups of animals

World association for the advancement of veterinary parasitology (WAAVP) guideline for the evaluation of the efficacy of anthelmintics in food-producing and companion animals: general guidelines

The general WAAVP (World Association for the Advancement of Veterinary Parasitology) guideline on anthelmintic efficacy were prepared to assist researchers with the planning, conduct and interpretation of studies to assess the efficacy of anthelmintic drugs in food-producing and companion animals. General principles are outlined herein to assist in the preparation and execution of dosage determination, dosage confirmation and field studies, which are applicable to all animal host species. These general guidelines are complemented by revised species-specific guidelines, which provide more specific, updated and detailed guidance for each animal host species

Exploring the Gastrointestinal “Nemabiome”: Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities

<div><p>Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions during co-infection, parasite epidemiology, parasite ecology and the response of parasite populations to both drug treatments and control programs.</p></div

Repeatability of the amplicon sequencing assay when applied to field samples.

<p>The amplicon sequencing assay was applied to DNA lysates made from populations of L3 larvae collected from field samples from six Canadian cattle farms (Field samples 1–6). <b>Fig 5A</b>: The results of three technical replicates of the amplicon sequencing assay applied to the same single DNA lysate made from larval populations of each farm. Each DNA lysate was made from 300–2000 larvae (Field sample 1 = 1000 L3; Field sample 2 = 400 L3; Field sample 3 = 1000 L3; Field sample 4 = 2000 L3; Field sample 5 = 300 L3 and Field sample 6 = 2000 L3. <b>Fig 5B</b>: The results of three technical replicates of the amplicon sequencing assay applied to three independent DNA lysates made from separate batches of 1,000 and 2,000 larvae from field samples 1 and 4 respectively. The Y-axis of both charts shows the percentage species proportions as determined by the amplicon sequencing assay (25 cycles of amplification and following application of the appropriate correction factor). The field sample number is indicated on the X-axis.</p

Comparison of the amplicon sequencing assay with visual morphological identification of larval populations from field samples.

<p>Fecal samples were collected from 39 individual calves entering feedlots from pasture at different locations in Oklahoma, Arkansas and Nebraska. 100 L3 larvae harvested from each coproculture were fixed and identified to the species level on the basis of morphological features on microscopy. The amplicon sequencing assay was applied to several hundred to several thousand L3 larvae from each sample. <b>Fig 6A</b>. Two bars are shown for each sample on the chart. The left bar of each sample shows the species proportions as determined by larval morphology. The right bar of each sample shows the proportions of each species as estimated by the amplicon sequencing assay (values shown are after application of the appropriate correction factor). The Y-axis shows the percentage proportions of each species as determined by morphology or the sequencing assay respectively. <b>Fig 6B</b>. Linear regression analysis was performed to assess the correlation between the percentage composition of each parasite species in each of 39 field samples as determined by the amplicon sequencing assay and morphological identification. The linear regression plots were produced using SPSS Statistics (IBM Corp. Released 2012. IBM SPSS Statistics for Macintosh, Version 21.0. Armonk, NY: IBM Corp), by plotting the percentage representation of each species from the morphological data (Y-axis) against the percentage representation of each species from the amplicon sequencing data (X-axis) for each sample. The values for R² (coefficient of determination), b (y-intercept) and m (slope) are shown adjacent to each plot. If values are the same the y-intercept (b) will be zero and the slope (m) will be 1.</p

The determination of species proportions of a series of pairwise parasite species combinations using the amplicon sequencing assay.

<p>DNA lysates were prepared from mock pools of 2000 L3 larvae comprising varying proportions of two different nematode species and the amplicon sequencing assay applied. Panel 3A, <i>O</i>. <i>ostertagi</i> and <i>H</i>. <i>placei</i>; Panel 3B, <i>C</i>. <i>oncophora</i> and <i>O</i>. <i>ostertagi</i>; Panel 3C, <i>O</i>. <i>ostertagi</i> and <i>C</i>. <i>punctata</i>; Panel 3D, <i>C</i>. <i>oncophora</i> and <i>C</i>. <i>punctata</i>. The left hand chart of each pair shows the expected results based on the known numbers of larvae added to each pool and the right hand chart of each pair shows the results of the amplicon sequencing assay (after the application of the appropriate correction factors). The ratios by which the two species of each pair vary from left to right on each chart are as follows: Column 1, 99:1; Column 2 90:10; Column 3; 70:30, Column 4, 50:50; Column 5; 30:70, Column 6; 10:90 and Column 7; 1:99.</p