Local immune response of Canarian Majorera goats infected with Teladorsagia circumcincta.

BACKGROUND: Due to increased anthelmintic resistance, alternative methods to drugs are necessary to control gastrointestinal nematodes (GINs). Some of the most promising alternatives are based on the immune response of the host, such as the selection of genetically resistant breeds or the use of vaccines against these parasites. Given the limited information available on the immune response against GINs in goats, this study investigated the local immune response of goat kids of an indigenous Canary Islands breed (Majorera breed) experimentally infected with Teladorsagia circumcincta, one of the most pathogenic and prevalent GIN species. METHODS: For this purpose, the relationship between different parasitological (number of mature and immature worms, worm length, and number of intrauterine eggs) and immunological parameters at the local level (related to both the humoral and cellular immune response) was analyzed at early (1 week post-infection [wpi]) and late (8 wpi) stages of infection. RESULTS: Primary infection of goat kids with T. circumcincta infective larvae (L3) generated a complex immune response that could be defined as Th2 type, characterized by increased infiltration in abomasal tissues of several effector cells as well as a progressive presence of specific antibodies against parasitic antigens in the gastric mucus. Cellular responses were evidenced from 1 wpi onward, showing an increase in antigen-presenting cells and various lymphocyte subsets in the gastric mucosa. CONCLUSIONS: The complexity of the host response was evidenced by statistically significant changes in the number of all these subpopulations (MHCII+, CD4+, CD8+, γδ+, CD45R+, IgA+, and IgG+), as well as in the evolution of the relative cytokine gene expression. From a functional point of view, negative associations were observed between the number of most of the immune cells (CD4, IgA, IgG, and CD45R cells) and parameters that could be related to the fecundity of worms, a phenomenon that was especially evident when the number of IgG and CD45R cells or the specific IgA levels of the gastric mucus were compared with parasitological parameters such as the female worm length or fecal egg counts at 8 wpi.

Regional heterogeneity and unexpectedly high abundance of Cooperia punctata in beef cattle at a northern latitude revealed by ITS-2 rDNA nemabiome metabarcoding.

BACKGROUND: The species composition of cattle gastrointestinal nematode (GIN) communities can vary greatly between regions. Despite this, there is remarkably little large-scale surveillance data for cattle GIN species which is due, at least in part, to a lack of scalable diagnostic tools. This lack of regional GIN species-level data represents a major knowledge gap for evidence-based parasite management and assessing the status and impact of factors such as climate change and anthelmintic drug resistance. METHODS: This paper presents a large-scale survey of GIN in beef herds across western Canada using ITS-2 rDNA nemabiome metabarcoding. Individual fecal samples were collected from 6 to 20 randomly selected heifers (n = 1665) from each of 85 herds between September 2016 and February 2017 and 10-25 first season calves (n = 824) from each of 42 herds between November 2016 and February 2017. RESULTS: Gastrointestinal nematode communities in heifers and calves were similar in Alberta and Saskatchewan, with Ostertagia ostertagi and Cooperia oncophora being the predominant GIN species in all herds consistent with previous studies. However, in Manitoba, Cooperia punctata was the predominant species overall and the most abundant GIN species in calves from 4/8 beef herds. CONCLUSIONS: This study revealed a marked regional heterogeneity of GIN species in grazing beef herds in western Canada. The predominance of C. punctata in Manitoba is unexpected, as although this parasite is often the predominant cattle GIN species in more southerly latitudes, it is generally only a minor component of cattle GIN communities in northern temperate regions. We hypothesize that the unexpected predominance of C. punctata at such a northerly latitude represents a range expansion, likely associated with changes in climate, anthelmintic use, management, and/or animal movement. Whatever the cause, these results are of practical concern since C. punctata is more pathogenic than C. oncophora, the Cooperia species that typically predominates in cooler temperate regions. Finally, this study illustrates the value of ITS-2 rDNA nemabiome metabarcoding as a surveillance tool for ruminant GIN parasites.

Variability in the Response against Teladorsagia circumcincta in Lambs of Two Canarian Sheep Breeds.

The increasing resistance to anthelmintics has necessitated the exploration of alternative control strategies of gastrointestinal nematode (GIN) infections. A sustainable option is genetic selection based on differences in susceptibility to GIN infection between and within breeds of sheep. Here, three-month-old Canaria Hair breed (GIN-resistant) and Canaria Sheep breed (GIN-susceptible) showed no significant between-breed differences after trickle infection with Teladorsagia circumcincta, whereas considerable individual variability was found in both breeds. Next, data from lambs of both breeds were used to explore the relationships between parasitological variables and T. circumcincta-specific IgA levels, local immune cell populations, and abomasal lymph node gene expression to understand the possible mechanisms underlying resistance. Mucosal IgA levels as well as numbers of globular leukocytes and MHC-II+ cells were associated with protection. Analysis of lymph node gene expression revealed the associations between lower parasite numbers and cumulative fecal egg counts and several immune pathways, such as leukocyte cell adhesion, activation and differentiation of T cells, in particular CD4+ and IL-4 production. The data obtained here may inform on the relationship between phenotypic resistance variability and protective responses at the humoral, cellular, and transcriptomic levels, thus contributing to identifying immune responses in young lambs that could be used as markers for selection.

Trichostrongyloid nematodes in ruminants of northern Iran: prevalence and molecular analysis.

BACKGROUND: This study was carried out to investigate the prevalence and analyze the molecular characteristics based on the internal transcribed spacer (ITS) 2 region of the ribosomal RNA (RNA) gene of trichostrongylid nematodes in different ruminants from Guilan province, northern of Iran. METHODS: The gastrointestinal tracts of 144 ruminants including 72 cattle, 59 sheep, and 13 goats were collected from an abattoir in Guilan province during July to September 2018. After isolation the helminths, male specimens were identified based on morphological parameters. PCR and partial sequencing of the ITS2 fragment were conducted. After phylogenetic analysis, the intraspecific and interspecific differences were calculated. RESULTS: The prevalence of total infections with the nematodes was 38.9, 74.6 and 84.6% among cattle, sheep and goats, respectively. Eleven species of trichostrongylid nematodes including Haemonchus contortus, Marshallagia marshalli, Trichostrongylus axei, T. colubriformis, T. vitrinus, Ostertagia trifurcata, Teladorsagia circumcincta, Marshallagia occidentalis, O. lyrata, O. ostertagi, and Cooperia punctate were recovered from the ruminants. The most prevalent trichostrongyloid nematodes in cattle, sheep and goats were O. ostertagi (26.4%), M. marshalli (64.4%) and T. circumcincta (69.2%), respectively. Phylogenetic tree was discriminative for Trichostrongylidae family, while phylogenetic analysis of the ITS2 gene represented low variations and no species identification of Haemonchidae and Cooperiidae families. CONCLUSIONS: This study suggests the high prevalence and species diversity of trichostrongyloid nematodes in different ruminants, indicating the importance of implement antiparasitic strategies in north regions of Iran. As well, this study showed that the ITS2 fragment is not a discriminative marker for Haemonchidae and Cooperiidae families, and investigation of other genetic markers such as mitochondrial genes would be more valuable for better understanding of their phylogenetic relationships.

The effect of Haemonchus contortus and Trichostrongylus colubriforms infection on the ruminal microbiome of lambs.

We evaluated Haemonchus contortus (HC) and Trichostrongylus colubriformis (TC) infection on the ruminal microbial community of Santa Ines lambs to better understand the pathophysiology of parasite infections and the interactions among gastrointestinal nematodes and gut resident microbiota. In this study, 18 six months of age lambs were maintained for 34 days in individual pens divided into three treatments that included animals infected with HC and TC, and control (infection-free). Haematological, ruminal parameter and microbial nitrogen absorbed by pune derivatives, as well as enteric methane emission (CH4), were analysed, and the rumen microbial taxonomic and functional profile assessed by shotgun metagenomics. The analysis showed that total protein, albumin, urea, and butyrate level were lower in animals infected by both parasites, while HC infection also decreased the haemoglobin level. Both infected groups (TC and HC) increased the enteric methane emission (CH4). TC and HC infections increased the diversity and richness of functional microbial genes. Most alterations in the rumen microbiome composition of infected groups are associated with the suppression of microbes involved in microbial homeostasis maintenance and expansion of the archaeal community in the infected animals. Infection led to an increased abundance of nitrogen, amino acid, protein, and energy metabolism genes. Overall, TC and HC infection increased the enteric methane emission, negatively affected taxon's responsible for maintenance de rumen homeostasis and modulated some important genes related to protein and energy metabolism.

New species of Hassalstrongylus (Trichostrongyloidea: Heligmonellidae) in the large-headed rice rat Hylaeamys seuanezi, in the Atlantic Forest of northeast Brazil.

A new species of Trichostrongyloidea (Nematoda: Heligmonellidae), Hassalstrongylus lauroi n. sp., is described from specimens collected from the small intestine of the rodent Hylaeamys seuanezi in the Atlantic Forest of northeastern Brazil (Igrapiúna, state of Bahia). The genus Hassalstrongylus includes 17 species, which parasitize rodents occurring in the Neotropical and Nearctic regions. It differs from the genus Stilestrongylus through its smaller number of ridges in the synlophe and through the size of the genital cone. The main taxonomic characteristics of this new species are the subsymmetrical caudal bursa of type 2-2-1, ray 8 branching out at the base of the dorsal trunk, right lobe smaller than the left, and rays 4 and 5 of robust nature. In addition, the ornamental ray 5 and the robustness of ray 4 on the male caudal bursa, along with the modification of the ridges of the posterior end of the female, allow us to consider the specimens found to be a new species.

Cellular and humoral immune responses associated with protection in sheep vaccinated against Teladorsagia circumcincta.

Due to increased anthelmintic resistance, complementary methods to drugs are necessary to control gastrointestinal nematodes (GIN). Vaccines are an environmentally-friendly and promising option. In a previous study, a Teladorsagia circumcincta recombinant sub-unit vaccine was administered to two sheep breeds with different levels of resistance against GIN. In the susceptible Canaria Sheep (CS) breed, vaccinates harboured smaller worms with fewer eggs in utero than the control group. Here, we extend this work, by investigating the cellular and humoral immune responses of these two sheep breeds following vaccination and experimental infection with T. circumcincta. In the vaccinated CS group, negative associations between antigen-specific IgA, IgG2 and Globule Leukocytes (GLs) with several parasitological parameters were established as well as a higher CD4+/CD8+ ratio than in control CS animals, suggesting a key role in the protection induced by the vaccine. In the more resistant Canaria Hair Breed (CHB) sheep the vaccine did not significantly impact on the parasitological parameters studied and none of these humoral associations were observed in vaccinated CHB lambs, although CHB had higher proportions of CD4+ and CD8+ T cells within the abomasal lymph nodes, suggesting higher mucosal T cell activation. Each of the component proteins in the vaccine induced an increase in immunoglobulin levels in vaccinated groups of each breed. However, levels of immunoglobulins to only three of the antigens (Tci-MEP-1, Tci-SAA-1, Tci-ASP-1) were negatively correlated with parasitological parameters in the CS breed and they may be, at least partially, responsible for the protective effect of the vaccine in this breed. These data could be useful for improving the current vaccine prototype.

Morphological and Molecular Data Reveal Two New Species of Viannaia (Nematoda: Viannaiidae), Parasitizing Opossums (Mammalia: Didelphidae) in Mexico.

Two new species of Viannaia from the intestine of the North American opossums, Didelphis virginiana (Virginia opossum), and Philander opossum (gray four-eyed opossum), are described based on morphological and molecular data, through an integrative taxonomic approach. Maximum likelihood and Bayesian inference analyses for each dataset and the concatenated dataset were performed using a mitochondrial cytochrome c oxidase subunit 1 (COI) gene, and the nuclear ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2). The phylogenetic analyses revealed 2 new species that occur in Mexico, one from the western state of Colima and another from the southern state of Chiapas. Our phylogenetic trees for both molecular markers and concatenated datasets yielded similar topologies with high bootstrap values and posterior probabilities. Viannaia is recovered as a monophyletic group, but the family Viannaiidae appears as non-monophyletic, due to the position of Travassostrongylus scheibelorum, similar to previous studies. Finally, the morphology of Viannaia and Hoineffia is discussed.

Vaccination against the brown stomach worm, Teladorsagia circumcincta, followed by parasite challenge, induces inconsistent modifications in gut microbiota composition of lambs.

BACKGROUND: Growing evidence points towards a role of gastrointestinal (GI) helminth parasites of ruminants in modifying the composition of the host gut flora, with likely repercussions on the pathophysiology of worm infection and disease, and on animal growth and productivity. However, a thorough understanding of the mechanisms governing helminth-microbiota interactions and of their impact on host health and welfare relies on reproducibility and replicability of findings. To this aim, in this study, we analysed quantitative and qualitative fluctuations in the faecal microbiota composition of lambs vaccinated against, and experimentally infected with, the parasitic GI nematode Teladorsagia circumcincta over the course of two separate trials performed over two consecutive years. METHODS: Two trials were conducted under similar experimental conditions in 2017 and 2018, respectively. In each trial, lambs were randomly assigned to one of the following experimental groups: (i) vaccinated/infected, (ii) unvaccinated/infected and (iii) unvaccinated/uninfected. Faecal samples collected from individual animals were subjected to DNA extraction followed by high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene and bioinformatics and biostatistical analyses of sequence data. RESULTS: Substantial differences in the populations of bacteria affected by immunisation against and infection by T. circumcincta were detected when comparing data from the two trials. Nevertheless, the abundance of Prevotella spp. was significantly linked to helminth infection in both trials. CONCLUSIONS: Despite the largely conflicting findings between the two trials, our data revealed that selected gut microbial populations are consistently affected by T. circumcincta infection and/or vaccination. Nevertheless, our study calls for caution when interpreting data generated from in vivo helminth-microbiome interaction studies that may be influenced by several intrinsic and extrinsic host-, parasite- and environment-related factors.

First report on parasites of European beavers in the Slovak Republic.

European beaver (Castor fiber L. 1758) is the biggest rodent living in Europe. It is a semi-aquatic animal known for building dams and burrows. European beaver is a potential host for a wide range of parasites and other infectious diseases. In Slovakia, there is an increasing number of beavers but the data about their parasitic fauna are missing. Our work is the first documentation about the beaver's parasitofauna in Slovakia. In a 1-year study, we collected and examined 19 beaver fecal samples from the vicinity of beaver burrows inhabiting three particular localities at the Danube, Topla, and Laborec rivers in Slovakia. In these fecal samples, 4 different species of intestinal endoparasites were detected as follows: oocysts of Cryptosporidium, cysts of Giardia, eggs of Stichorchis subtriquetrus, and eggs and larvae of Travassosius rufus. Parasites were confirmed only in samples collected at river Topla. Based on our results, we can conclude that European beaver can be an important source of parasitic contamination of surface waters especially in the localities shared by people.

Differences within Churra breed sheep in the early immune response to the infection by Teladorsagia circumcincta.

This study describes early immunological mechanisms that underlie resistance to Teladorsagia circumcincta infection in adult Churra sheep. After a first experimental infection, 6 animals were classified as resistant (RG) and 6 as susceptible (SG) to T. circumcincta infection based on their cumulative faecal egg count (cFEC) at the end of the infection. RG showed higher IgA levels against somatic antigen of T. circumcincta fourth-larvae stage (L4) in serum at day 3 post-infection (pi) (p < 0.05) and close to significance at day 21 pi (p = 0.06). Moreover, a strong negative correlation between cFEC and specific IgA was only significant in RG at day 3 pi (r = - 0.870; p < 0.05), but absent in SG. At the end of this infection, sheep were treated with moxidectin and infected again 3 weeks later to be slaughtered at day 7 pi. At necropsy, the specific IgA levels in gastric mucosa were similar between groups; the absence differences at day 7 pi could be due to a previous increase in the IgA response, probably around day 3 pi, as described during the first infection. L4 burden, 68% lower in RG than in SG, was influenced by the specific IgA in gastric mucus and the number of γδ T cells. RG group showed a positive correlation between γδ T cells and eosinophils (r = 0.900; p = 0.037); however, this correlation was not found in SG. These results show that these two phenotypes show different early immune response pattern to T. circumcincta infection in Churra sheep.

New species of Hassalstrongylus (Trichostrongyloidea: Heligmonellidae) in the large-headed rice rat Hylaeamys seuanezi, in the Atlantic Forest of northeast Brazil / Nova espécie de Hassalstrongylus (Trichostrongyloidea: Heligmonellidae) no roedor Hylaeamys seuanezi, na Mata Atlântica do nordeste do Brasil

Abstract A new species of Trichostrongyloidea (Nematoda: Heligmonellidae), Hassalstrongylus lauroi n. sp., is described from specimens collected from the small intestine of the rodent Hylaeamys seuanezi in the Atlantic Forest of northeastern Brazil (Igrapiúna, state of Bahia). The genus Hassalstrongylus includes 17 species, which parasitize rodents occurring in the Neotropical and Nearctic regions. It differs from the genus Stilestrongylus through its smaller number of ridges in the synlophe and through the size of the genital cone. The main taxonomic characteristics of this new species are the subsymmetrical caudal bursa of type 2-2-1, ray 8 branching out at the base of the dorsal trunk, right lobe smaller than the left, and rays 4 and 5 of robust nature. In addition, the ornamental ray 5 and the robustness of ray 4 on the male caudal bursa, along with the modification of the ridges of the posterior end of the female, allow us to consider the specimens found to be a new species.

Resumo Uma nova espécie de Trichostrongyloidea (Nematoda: Heligmonellidae), Hassalstrongylus lauroi n. sp., descrita a partir de espécimes coletados do intestino delgado do roedor Hylaeamys seuanezi na Mata Atlântica do Nordeste do Brasil (Igrapiúna, estado da Bahia). O gênero Hassalstrongylus inclui 17 espécies, que parasitam roedores que ocorrem nas regiões Neotropical e Neártica. Este difere do gênero Stilestrongylus por ter menor número de cristas na sínlofe e pelo tamanho do cone genital. As principais características taxonômicas dessa nova espécie são a bolsa caudal subsimétrica do tipo 2-2-1, raio 8, ramificando-se na base do tronco dorsal, lobo direito menor que o esquerdo e os raios 4 e 5 são robustos. Além disso, o raio 5 é ornamentado na bolsa caudal dos machos, e ocorre uma modificação nas cristas da extremidade posterior da fêmea, que permite considerar os espécimes encontrados como uma espécie nova.

Within-host mechanisms of immune regulation explain the contrasting dynamics of two helminth species in both single and dual infections.

Variation in the intensity and duration of infections is often driven by variation in the network and strength of host immune responses. While many of the immune mechanisms and components are known for parasitic helminths, how these relationships change from single to multiple infections and impact helminth dynamics remains largely unclear. Here, we used laboratory data from a rabbit-helminth system and developed a within-host model of infection to investigate different scenarios of immune regulation in rabbits infected with one or two helminth species. Model selection suggests that the immunological pathways activated against Trichostrongylus retortaeformis and Graphidium strigosum are similar. However, differences in the strength of these immune signals lead to the contrasting dynamics of infections, where the first parasite is rapidly cleared and the latter persists with high intensities. In addition to the reactions identified in single infections, rabbits with both helminths also activate new pathways that asymmetrically affect the dynamics of the two species. These new signals alter the intensities but not the general trend of the infections. The type of interactions described can be expected in many other host-helminth systems. Our immune framework is flexible enough to capture different mechanisms and their complexity, and provides essential insights to the understanding of multi-helminth infections.

Purpureocillium lilacinum and Trichoderma virens for biological control of trichostrongylid parasites of sheep: an in vitro evaluation.

The study evaluated the ovicidal activity of enzymatic extracts of Purpureocillium lilacinum and Trichoderma virens against trichostrongylid eggs from sheep. Filtered extract (FE) and macerated crude extract (MCE) were prepared from fungal cultures in minimal broth. In the experiment, 100 trichostrongylid eggs, obtained from the feces of naturally infected sheep, were exposed to fungal extracts for 24 and 48 hours/25°C. In the control group, eggs were incubated in minimal broth. The number of L1 larvae was ascertained. Each treatment consisted of four repetitions and the experiment was repeated five times. It was observed that the effect of FE and MCE of P. lilacinum and T. virens on egg hatchability differed from that of the control group. MCE of T. virens and P. lilacinum showed higher ovicidal activity than FE over both periods and at 48 hours of exposure, respectively. From the percentage reductions in hatchability of the eggs, MCE was shown to be superior to FE for both fungi. This study demonstrated the ovicidal potential of these fungi against trichostrongylid eggs. However, further studies are needed in order to identify the molecules responsible for the ovicidal effects, and to evaluate the behavior of fungal extracts in biotic and abiotic interactions.

Parasite intensity drives fetal development and sex allocation in a wild ungulate.

An understanding of the mechanisms influencing prenatal characteristics is fundamental to comprehend the role of ecological and evolutionary processes behind survival and reproductive success in animals. Although the negative influence of parasites on host fitness is undisputable, we know very little about how parasitic infection in reproductive females might influence prenatal factors such as fetal development and sex allocation. Using an archival collection of Dall's sheep (Ovis dalli dalli), a capital breeder that depends on its body reserves to overcome the arctic winter, we investigated the direct and indirect impacts of the parasite community on fetal development and sex allocation. Using partial least squares modelling, we observed a negative effect of parasite community on fetal development, driven primarily by the nematode Marshallagia marshalli. Principal component analysis demonstrated that mothers with low parasite burden and in good body condition were more likely to have female versus male fetuses. This association was primarily driven by the indirect effect of M. marshalli on ewe body condition. Refining our knowledge of the direct and indirect impact that parasite communities can have on reproduction in mammals is critical for understanding the effects of infectious diseases on wildlife populations. This can be particularly relevant for species living in ecosystems sensitive to the effects of global climate change.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance.

BACKGROUND: Benzimidazole resistance is associated with isotype-1 ß-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 ß-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Host specificity and phylogeny of Trichostrongylidae of domestic ruminants in the Guinea savannah of the Adamawa plateau in Cameroon.

Gastro-intestinal tracts were examined from thirteen Gudali zebu cattle, ten goats and ten sheep from the Adamawa highland in Northern Cameroon. A total of 28,325 adult helminths were recovered from the abomasa, small and large intestines. Five trichostrongylid genera were identified by their morphology: Haemonchus, Trichostrongylus and Oesophagostomum were predominant in both cattle and small ruminants, whilst Cooperia was only found in cattle both in the abomasum and small intestines. The molecular species identification and the inference of their phylogenetic relationships was based on the analysis of the hypervariable region I of the small subunit 18S rDNA (SSU) and the Second Internal Transcribed Spacer (ITS-2) of 408 adult trichostrongylid worms, which were PCR-amplified, sequenced, and compared with available database entries. Consistent with earlier findings, the SSU was invariable within the Haemonchus and Trichostrongylus genera, confirming the prior classification based on the morphology of the worms, but the ITS-2 was highly inter- and intraspecifically variable and thus allowed to distinguish individual species and to study the haplotype diversity within the different species. In cattle, we report for the first time in Cameroon co-infection with two species of Haemonchus (H. placei and H. similis), together with two species of Cooperia (C. punctata and C. pectinata) and one species of Trichostrongylus (T. axei). In goats and sheep, we found one highly polymorphic clade of Haemonchus contortus and two Trichostrongylus species (T. axei and T. colubriformis). When compared with other Trichostrongylidae from different regions of the world and wildlife, the analysis of haplotypes did not indicate any host and geographical isolation, but a very high haplotype diversity among H. contortus. These findings illustrate the complexity of trichostrongylid populations in domestic ruminants and suggest grazing overlap between domestic and wildlife hosts.

Motility based assays using cultured fourth stage larvae fail to provide consistent discrimination between known avermectin-resistant and -susceptible isolates of Cooperia spp.

The fecal egg count reduction test (FECRT) is the only method commonly used for diagnosing anthelmintic resistance in gastrointestinal nematodes of cattle, but this method has several drawbacks that have limited its widescale implementation. Consequently, there exists a need to develop better methods for diagnosing resistance. Assays based on larval motility are used commonly for screening potential drug candidates, and for detecting drug resistance, but previous work in our lab demonstrated that the L3 stage failed to discriminate between avermectin-resistant and susceptible isolates of Cooperia spp. We hypothesized that the L4 may be a better stage for this purpose because it is a parasitic and actively feeding life stage without a double cuticle. L3 larvae of Cooperia spp. were exsheathed and cultured to L4 by maintaining them in media at 37 °C and 20 % CO2, with media changes and observation every 48 h for nine days. Three avermectin-resistant and two avermectin-susceptible GIN isolates (diagnosed by FECRT) containing >88 % Cooperia spp., were used. Three biological replicates were performed for each parasite isolate using both eprinomectin and ivermectin. Eleven drug concentrations from 0.01um to 40um and negative controls were evaluated. Motility readings were taken using the Worminator system before addition of the drug and at 24- and 48 -hs post drug exposure. Resistance ratios for ivermectin and eprinomectin ranged from 0.35 to 2.75 and 0.54-1.03, respectively. Though significant differences (p < 0.05) in percent inhibition were found at some drug concentrations in some assays, there were no consistent significant differences in the dose-response between susceptible and resistant isolates. Inhibition was greater in about half of the assays for the susceptible isolates, and in half the assays for the resistant isolates. The lack of consistency in these data indicate that motility of L4 is not a reliable diagnostic phenotype for measuring resistance to avermectin drugs in Cooperia spp.

Efficacy and production benefits following use of eprinomectin extended-release injection in pastured dairy heifers.

A study was conducted in grazing dairy heifers to assess anthelmintic efficacy and production responses in dairy heifers treated with a single injection of eprinomectin in an extended-release formulation over a 123 day-period. The study was conducted on a pasture-based dairy in the Southeastern United States (North Carolina) over the summer months. Sixty crossbred dairy heifers were weighed and randomly allocated into 2 groups. One group (n = 30) was given 5% eprinomectin subcutaneously in the cervical region while the other group (n = 30) was given an equivalent volume of saline. Calves were weighed every 30 days throughout the trial for calculation of average daily gain and differences in overall weight gain. In addition, fecal samples were collected at days 0, 30, 60, 90 and 123 for worm egg count and coproculture. Both groups of cattle had similar worm egg concentrations at the start of the study. However, the control group had increasing concentrations of fecal worm eggs throughout the summer months while the heifers that received eprinomectin had minimal fecal worm eggs. The primary parasite species identified in this study were Haemonchus placei, Cooperia species and Ostertagia. The heifers that received eprinomectin gained 105 + 2.8 kg during the 123-day study period, representing an average daily gain of 0.85 kg/day compared to 78.3 + 4.1 kg (0.64 kg/day) for the control group. This represented a 33 % increase in average daily gain associated with deworming. The results of this study indicate that a single dose of extended-release eprinomectin was sufficient to control parasites through a 123-day summer grazing season and that administration of the anthelmintic had a significant impact on weight gain.

High frequency of benzimidazole resistance alleles in trichostrongyloids from Austrian sheep flocks in an alpine transhumance management system.

BACKGROUND: Infections of small ruminants with trichostrongyloid nematodes often result in reduced productivity and may be detrimental to the host. Anthelmintic resistance (AR) against most anthelmintic drug classes is now widespread amongst the trichostrongyloids. Baseline establishment, followed by regular monitoring of the level of AR, is necessary for farmers and veterinarians to make informed decisions about parasite management. The detection of single nucleotide polymorphisms (SNPs) is a sensitive method to detect AR against benzimidazoles (BZs), one of the most widely used anthelmintic classes. Alpine transhumance constitutes a special type of pasturing of sheep from many different farms, the aim of this study was to investigate the prevalence of benzimidazole resistance alleles in this particular management system. RESULTS: Sixteen sheep flocks in Styria and Salzburg in Austria were examined by pyrosequencing for SNPs at codons 167, 198 and 200 of the isotype-1 ß-tubulin gene. The frequency of the resistance-associated exchange F200Y was 87-100% for H. contortus, 77-100% for T. colubriformis and <  5-66% for T. circumcincta. Additionally, the F167Y polymorphism was detected in T. colubriformis from two farms at a frequency of 19 and 23% respectively. CONCLUSIONS: The high resistance allele frequency in H. contortus and T. colubriformis in the examined sheep population urgently calls for the development of new treatment strategies to sustainably control trichostrongyloid infections for this kind of pasturing, since the frequent mixing of flocks during the alpine summer grazing must be considered an important risk factor for the spread of resistant nematodes to a large number of farms.