Nematodes In Cattle Research Articles

201 Transcriptome analysis of bovine abomasal mucosa following experimental infection with Ostertagia ostertagi

Abstract Ostertagia ostertagi is a cattle stomach nematode that causes significant disease; however, little is known about host responses to infection. Due to the emergence of drug resistance in these parasites, there is a need for alternative treatments. To understand host responses to O. ostertagi infection in the gastric mucosa, gene expression in fundic and pyloric mucosa was compared between infected and uninfected calves for 21 d post-infection. The number of differentially expressed genes (DEGs) increased over time with more upregulated than downregulated DEGs at each time point. Fundic mucosa had more DEGs than pyloric mucosa across all time points. The largest changes occurred between d 7 to 9 and 10 post-infection. Most DEGs are associated with immunity, cellular reorganization, cell migration, and proliferation. Tuft/epithelial cell response to the infection was atypical, lacking key cytokines. Numerous genes associated with T cell exhaustion were upregulated. The data collectively indicate that O. ostertagi infection elicited host immune responses, yet also suggest parasite-induced immunosuppression is present. This may explain why cattle are slow in developing protective immunity to O. ostertagi. Understanding mechanisms of parasite evasion will facilitate the rational design of protective vaccines against complex nematode parasites.

Comprehensive approach to in silico identification and in vitro validation of anti-filarial hit molecules targeting the dimer interface of thioredoxin peroxidase 1 in Wuchereria bancrofti: a progress in anti-filariasis drug development.

Lymphatic filariasis (LF) remains a significant health challenge for populations in developing countries. LF is a parasitic disease transmitted by mosquitoes, mainly caused by the filarial nematode, Wuchereria bancrofti, prevalent in tropical and subtropical regions. Since the present drugs develop complications, including adverse side effects, lack of specificity, and development of drug resistance, the present study focused on developing the potential anti-filariasis drugs targeting crucial proteins for the nematode life cycle. We have identified the therapeutic compounds by targeting the enzyme thioredoxin peroxidase 1 (WbTPx1), which facilitates the conversion of hydrogen peroxide into water, an essential mechanism by which the nematode survives against oxidative stress in the host. This approach might resolve treatment efficacy and activity difficulties at various stages of filarial parasitic worms. We modeled the structure of WbTPx1 and employed the structure-based virtual screening approach, focusing on the dimer interface region of the protein. ADMET prediction profiles of the non-toxic, top-ranked hits with higher docking scores demonstrate higher affinity to the nematode protein than its human homolog. The molecular dynamic simulation studies show WbTPx1-hit complexes' stability and the intactness of hits in the binding site. Further, in vitro validation of identified hits using Setaria digitata, a cattle nematode, showed better IC50 and higher inhibition than the standard drug ivermectin, indicating the potential to inhibit enzyme activity and the development of drug candidates for controlling LF.

Biomarkers of gastrointestinal nematodes in beef cattle raised in a tropical area.

Biomarkers are specific molecular, histological, or physiological characteristics of normal or pathogenic biological processes and are promising in the diagnosis of gastrointestinal nematodes (GINs). Although some biomarkers have been validated for infection by Ostertagia sp. in cattle raised in temperate regions, there is a lack of information for tropical regions. The aim of this project was to assess potential biomarkers and validate the most promising. In the first study, 36 bovines (Nelore breed) naturally infected by GINs were distributed into two groups: infected (not treated with anthelmintic) and treated (treated with fenbendazole on days 0, 7, 14, 21, 28, 42, and 56). The variables of interest were live weight, fecal egg count, hemogram, serum biochemical markers, phosphorus, gastrin, and pepsinogen. In the second step, pepsinogen was assessed in cattle of the Nelore breed distributed among three groups: infected (not treated with anthelmintic), MOX (treated with moxidectin), and IVM + BZD (treated with ivermectin + albendazole). In the first study, no difference between groups was found for weight, albumin, hematocrit (corpuscular volume [CV]), erythrocytes, or hemoglobin. Negative correlations were found between pepsinogen and both CV and albumin, and albumin was negatively correlated with the percentage of Haemonchus sp. in the fecal culture. Among the biomarkers, only pepsinogen differentiated treated and infected (beginning with the 28th day of the study). In the second study, a reduction in pepsinogen was found after anthelmintic treatment. Therefore, pepsinogen is a promising biomarker of worms in cattle naturally infected by the genera Haemonchus and Cooperia in tropical areas.

Microscopic and molecular epidemiology of gastrointestinal nematodes in dairy and beef cattle in Pak Chong district, Nakhon Ratchasima province, Thailand.

Gastrointestinal (GI) nematode infection remains an important problem in livestock, particularly cattle. The infection may lead to serious health complications and affect animal products. The objective of this study was to investigate GI nematode infection and its associated risk factors in dairy and beef cattle farmed in Pak Chong District of Nakhon Ratchasima Province, northeast Thailand. Fecal specimens were collected from 101 dairy cattle and 100 beef cattle. Formalin-ethyl acetate concentration techniques were used to process the samples and the samples were observed under a light microscope. Samples were subjected to molecular identification of specific genera using conventional polymerase chain reaction and DNA sequencing. The overall prevalence of GI nematode infection was 33.3%. The strongyle nematode was the most significant GI nematode in this area with a prevalence of 28.4%. The prevalence of strongyle nematodes was 58.0% in beef cattle and only 7.9% in dairy cattle. Trichuris spp. was another nematode found in both types of cattle with an overall prevalence of 5.0% with 9.0% in beef cattle and 1.0% in dairy cattle. The results of the epidemiological study indicate that the age of cattle, food, water sources, farming system, and housing floor are the most important risk factors. Among the strongyle nematodes, Ostertagia spp. was the most prevalent (82.0%), followed by Haemonchus spp. (62.3%) and Trichostrongylus spp. (8.2%), respectively. Infection with GI nematodes still exists in this area, particularly in beef cattle. Our reported data may benefit local parasitic control policies in the future.

Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

EPG and Pasture Larval Count of Gastrointestinal Nematodes are Strongly Influenced by Weather Parameters: Empirical Evidence from Parbhani Marathwada

Aims: the present study was aimed to develop a bioclimato of gastrointestinal nematodes of cattle in Marathwada region of Maharashtra. Study Design: A standard protocol as per published reports was followed for recording of prevalence of GI nematodes from cattle. Place and Duration of Study: The study was conducted at livestock instructional farm of College of Veterinary and Animal Sciences Parbhani during the period February 2022 to January, 2023. Methodology: In all total 253 faecal samples were collected from calves and adult cattle belonging to RK instructional farm, COVAS, Parbhani. Sample collection and processing was done by following standard parasitological procedures and only fresh fecal droppings were used for the laboratory investigation. Results: The study conducted on availability of larvae on pasture revealed the overall prevalence of 61 percent (87 pasture were positive out of 143 examined). The PLC analysis showed the highest distribution of larvae on pasture during winter season i.e. 57.81 %, followed by monsoon season i.e. 57.81 % and 00.0% during summer season. The Pasture larval count observed during different seasons was 23.83, 33.07 and 0.00 per 100 gm, respectively. The abstract information drawn from the bioclimatos plotted for 2000-2021 and its validation with real time data of 2022 ( Feb 2022-Jan 2023) was a) Suitable months for survival of Heamonchus and Oesophagostomum infective larvae on the pasture of the grazing land in year 2022-2023 were Jan - March and June – September and for Trichostrongylus Jan – March and October 2022- January 2023 and b)The climatic data and Bioclimato were plotted from year 2000-2021 and 2022+January 2023 showed the correlation between EPG and PLC. Conclusion: The bioclimatos can be developed and utilized according the regional agro-climatic variations and be useful for devising controlmeasures against gastrointestinal nematodes of livestock.

Environmental Factors Influence the Infestation of Gastrointestinal Nematodes in Cattle in Aceh Province

Environmental Factors Influence the Infestation of Gastrointestinal Nematodes in Cattle in Aceh Province

Ovicidal Effect of Disinvasive Substances on the Eggs of Nematodiroses Causative Agents

The article deals with the study of the disinvasive effects of various percentage solutions of chemical substances on the eggs of nematodes in cattle under in vitro conditions. Based on the results obtained during the experiments, carried out under in vitro conditions it was found that 3.0% and 5.0% solutions of the deltatrine preparation destroy nematode eggs by 100%, and 3.0% and 5.0% solutions of the citrine preparation by 72.2-80.0%. In a concrete test object, with a 3-hour exposure, helminth eggs (1200 eggs) were destroyed by 100% from the impact of a 3.0% deltathrine preparation, by 63.6% from the impact of a 3.0% citrine solution (800 eggs), and by 58.3% from the impact of 4.0% phenol (700 eggs). Also on a wooden test object, with a 3-hour exposure, helminth eggs (1200 eggs) were destroyed by 100% from the impact of 3.0% deltathrine preparation, by 58.3% from the impact of a 3.0% citrine (700 eggs), and by 63.6% from the impact of 4.0% phenol solutions (600 eggs). For control purposes, ordinary water was added to the helminth eggs, and no decrease in their number was detected. At the next stage, having isolated them from other areas, 4 plots of 1 m2 each were allocated. For the purpose of the experiment, 93.3% of eggs died on a plot sprayed with a 3.0% solution of the deltatrine preparation, 66.7% of eggs died on a plot sprayed with a 3.0% solution of the citrine preparation, and 60.0% of eggs died on a plot sprayed with a 4.0% solution of phenol. Since under in vitro conditions, due to the direct effect of disinvasive substances on Nematodirus eggs, the eggs die quickly, however, in test objects, due to the fact that the eggs are mixed with feces on an area of 1 m2, the duration of action of chemicals increases, and the percentage of destruction of helminth eggs decreases.

The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019–2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

Ecology of the free-living stages of cattle nematodes in the dry season in the Lerma Valley, Salta province, Argentina

The objective of this work was to describe the dynamics of development and survival of the free-living stages of cattle gastrointestinal nematodes (GIN) in fecal matter (FM) and pasture during the dry season in the Lerma Valley, Salta province, northwestern Argentina (NWA) to contribute to GIN management. The climate in the region is characterized by a rainy summer followed by a dry season from middle autumn to early spring. Fecal matter from calves naturally infected with GIN was deposited on three experimental field plots in April, July and October 2019, corresponding to the beginning, middle and end of the dry season, respectively. Each experimental unit consisted of 7 stools of about 800 g and had four repetitions. To determine the development from egg to infective larvae (L3), the first sampling (5 g fecal matter) was performed from the 10th day post-contamination and continued every 3 days until L3 were found. Subsequently, a monthly sampling was made until two consecutive negative results were obtained. Sampling of pasture began three days after the L3 recovery from FM, and continued monthly until two negative results were obtained. The following parameters were evaluated: development time and development rate from egg to L3; permanence time of L3 in feces; time of appearance on pasture; migration rate; and permanence time of L3 on pasture. The main genera of parasites present were Cooperia and Haemonchus. Significant differences were observed in the development time among contamination months (p < 0.001); development time was highest in the July contamination (28 days), with October and April contamination averaging 9 and 10 days, respectively. Development time also showed significant differences (p < 0.01) among contamination months, being highest in October (31.48%). The highest permanence time in fecal matter values were recorded in the July contamination (183 days) and migration rate was highest in the October contamination (42.49%). The highest time of appearance on pasture value was recorded in the July contamination (117 days). Finally, the highest permanence time of L3 in feces values were detected in the October contamination (148 days). The results of this work show that fecal contamination in the NWA region in the dry season would play an epidemiological role in the GIN cycle as a source of infection for the next productive cycle in the rainy season.

Determination of prevalence and associated risk factors of Gastrointestinal Nematodes in Cattle at Sylhet Region, Bangladesh

Determination of prevalence and associated risk factors of Gastrointestinal Nematodes in Cattle at Sylhet Region, Bangladesh

Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP.

The bovine lungworm, Dictyocaulus viviparus (Bloch, 1782), is highly pathogenic and disease outbreaks can be difficult to predict and manage. Rapid and accurate diagnosis is vital, but without a sensitive diagnostic test this remains challenging in clinical practice. High performance molecular detection tools are therefore required to improve the diagnosis of this parasite and promote the implementation of strategic control measures. Loop-mediated isothermal amplification (LAMP), a rapid DNA assay, offers potential for field-based detection. Here we report a novel LAMP assay (DviLAMP), that was designed to target the D. viviparus internal transcribed spacer 2 (ITS2) ribosomal DNA region. Firstly, genomic DNA was extracted from a single D. viviparus L1 larva to amplify and clone the ITS2 into the recombinant plasmid (DviITS2). The DviLAMP successfully detected the target, with results shown by gel electrophoresis and real-time analysis, in addition to point-of-care amenable end-point detection: colorimetry and lateral flow dipstick (LFD). Analytical sensitivity can detect 0.5 ng DviITS2 following 45 min of incubation at 64°C, increasing to just 1 pg following 90 min of incubation. Using the same primers, other nematodes of cattle, Ostertagia ostertagi and Cooperia oncophora, were also detectable both by gel electrophoresis and real-time. However, when FITC and biotin tagged primers were incorporated to adapt the DviLAMP to LFD end-point detection, the LFD showed specific detection of D. viviparus. Further development of DviLAMP as a point-of-care test could significantly improve the sensitivity of lungworm diagnosis in the field.

Automating parasite egg detection: insights from the first AI-KFM challenge.

In the field of veterinary medicine, the detection of parasite eggs in the fecal samples of livestock animals represents one of the most challenging tasks, since their spread and diffusion may lead to severe clinical disease. Nowadays, the scanning procedure is typically performed by physicians with professional microscopes and requires a significant amount of time, domain knowledge, and resources. The Kubic FLOTAC Microscope (KFM) is a compact, low-cost, portable digital microscope that can autonomously analyze fecal specimens for parasites and hosts in both field and laboratory settings. It has been shown to acquire images that are comparable to those obtained with traditional optical microscopes, and it can complete the scanning and imaging process in just a few minutes, freeing up the operator's time for other tasks. To promote research in this area, the first AI-KFM challenge was organized, which focused on the detection of gastrointestinal nematodes (GINs) in cattle using RGB images. The challenge aimed to provide a standardized experimental protocol with a large number of samples collected in a well-known environment and a set of scores for the approaches submitted by the competitors. This paper describes the process of generating and structuring the challenge dataset and the approaches submitted by the competitors, as well as the lessons learned throughout this journey.

Plant-based production of a protective vaccine antigen against the bovine parasitic nematode Ostertagia ostertagi

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes.

Dose confirmation of a novel fixed dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) against naturally acquired gastrointestinal nematodes in US cattle

Macrocyclic lactone (ML) resistance in cattle gastrointestinal nematodes (GINs) is an increasing problem. Concurrent combination anthelmintic therapy incorporating an existing ML with a second drug class has been proposed to control cattle GINs while slowing the development of ML resistance. Two dose confirmation studies were conducted to investigate the efficacy of a new fixed-dose combination injectable (FDCI) anthelmintic against common cattle GINs known to negatively impact production. The FDCI is formulated with 5 mg/ml doramectin and 150 mg/ml levamisole hydrochloride (HCl). Cattle enrolled in the two studies were sourced from either the Southern (Study 1, n = 30) or Midwest (Study 2, n = 36) United States. Animals with GIN infections confirmed by fecal egg count (FEC) were randomly allocated to one of three treatment groups. On Day 0, cattle with positive FECs on Day −5( ± 2) were weighed and administered a single subcutaneous injection of either saline (0.9% sodium chloride) at 0.04 ml/kg, 10 mg/ml doramectin at 0.02 ml/kg (to provide 0.2 mg/kg doramectin) or the FDCI at 0.04 ml/kg (to provide 0.2 mg/kg doramectin and 6.0 mg/kg levamisole HCl). On Day 14, fecal samples were collected, animals were euthanized, and worms were collected from the intestinal tract of each animal. Treatment efficacy was calculated using worm burdens and the fecal egg count reduction test (FECRT). Pre-treatment (Day −5, Study 1; Day −3, Study 2) mean FECs were 999.4–1136.2 eggs per gram (EPG) in Study 1 and 137.1–226.6 EPG in Study 2. The FDCI was active against cattle GIN populations in both studies, with FECRT ≥ 99.98% in both studies. Compared to saline-treated cattle, FDCI-treated cattle had significantly fewer adult and immature worms of all identified species on Day 14. In Study 1, Day 14 efficacy of the FDCI was 96.9% for Cooperia spp. (C. oncophora (99.7%) and C. punctata (95.9%)), 99.1% for Nematodirus helvetianus, and 99.8% for Ostertagia spp. In Study 2, the FDCI provided 100% efficacy against all adult GIN species identified, including all GINs identified in Study 1 and Trichostrongylus axei. The FDCI also provided 95.5% efficacy against immature Ostertagia spp. and 100% efficacy against immature Cooperia spp. (Study 2). Doramectin was effective against all adult cattle GINs (except N. helvetianus) in Study 2 but was only effective against adult Ostertagia spp. in Study 1. Additionally, doramectin was only effective against immature Cooperia spp. (and not immature Ostertagia spp.) in Study 2. A single administration of the doramectin + levamisole HCl FDCI provides a new and effective approach to the treatment and control of common cattle GINs, including those exhibiting decreased susceptibility to doramectin alone.

Efficacy of a fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) in Australian cattle against naturally acquired gastrointestinal nematode infections

Australian producers have long used macrocyclic lactones (MLs) to successfully control cattle gastrointestinal nematodes (GINs) and consequently improve production parameters. However, the trajectory of ML resistance development in cattle GINs is following that of small ruminant nematode populations, highlighting a need for novel treatment options to provide efficacy in the current environment and interrupt the long-term establishment of ML-resistant GIN populations in Australian cattle. Here, we describe three field studies conducted in Australia to evaluate the efficacy of a single administration of a novel fixed-dose combination injectable (FDCI) endectocide against naturally acquired infections of cattle GINs. The FDCI is administered subcutaneously to deliver 0.2 mg/kg doramectin and 6 mg/kg levamisole hydrochloride (HCl). Study sites consisted of three farms in New South Wales (n = 2) and Victoria (n = 1). At each site, cattle were randomly allocated into one of three treatment groups: (1) untreated control (saline), (2) FDCI (0.2 mg/kg doramectin, 6 mg/kg levamisole HCl) or (3) positive control (0.2 mg/kg ivermectin). All treatments were administered on Day 0. Fecal samples were collected prior to treatment on Days −1 (Study 3) or 0 (Studies 1 and 2) and again on Day 14 (post-treatment) to evaluate efficacy via fecal egg count (FEC) and for coproculture. Adequacy of infection was confirmed at all three study sites, with Day 14 geometric mean (GM) FECs for saline-treated cattle ranging from 32.5 eggs per gram (EPG) to 623.7 EPG. FECs for FDCI-treated cattle were significantly reduced compared to saline-treated cattle (p ≤ 0.0001) on Day 14, with GM-based efficacy ≥ 99.7% at all three study sites. In contrast, ivermectin was 97.4% effective against cattle GINs in Study 1 but was only 47.2% and 39.8% effective at study site 2 and 3, respectively. Genus-specific efficacies suggest the presence of ivermectin-resistant Cooperia spp. (Study 1), Haemonchus spp. (Study 2) and Ostertagia spp. (Study 3) populations in the naturally infected cattle used in these studies. The post-treatment FEC and genus-specific efficacy estimations indicate the doramectin + levamisole HCl FDCI was highly efficacious against cattle GINs even in the face of ivermectin LOE at study sites 2 and 3. The efficacy of the new FDCI against both ML-susceptible and ML-resistant economically important cattle GINs in Australia affirms it is a valuable treatment option for producers operating in an environment of ML loss of efficacy.

Reassessing Stephanofilaria stilesi dermatitis in cattle, with characterization of molecular markers for confirming diagnosis

BackgroundStephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans) intermediate host. Despite being relatively common, little attention has been given to a thorough description of S. stilesi lesions and the potential integration of pathological and molecular diagnostic findings to confirm infection.MethodsTo characterize the cutaneous lesions caused by S. stilesi in cattle (Bos taurus taurus and Bos taurus indicus), skin of the ventral abdominal midline was collected from 22 animals during postmortem examination. Skin samples were processed for histology, transmission electron microscopy (TEM), DNA extraction, PCR, and Sanger sequencing targeting molecular markers cytochrome oxidase c subunit 1 (cox1), 12S, 18S rDNA, and 28S rDNA.ResultsMacroscopically, lesions ranged from 5 × 4 cm to 36 × 10 cm, consisting of one large single lesion, or two to four ovoid areas at the ventral abdominal midline, surrounding the umbilicus. Each lesion presented as ulcerative dermatitis with dry, serocellular crusts, or alopecic and lichenified areas. Histologically, eosinophilic, neutrophilic, and ulcerative dermatitis with furunculosis, folliculitis, and epidermal hyperplasia was observed. Cross sections of adult nematodes were identified in ~ 60% of the cases (n = 13) within intact follicles, sebaceous ducts, crusts, and areas of furunculosis. Stephanofilaria first-stage larvae (L1) were observed in five cases within “vitelline membranes” in the superficial dermis and crusts. Ultrastructurally, the L1 cross sections were compounded of smooth multilayered cuticle and somatic cells. The “vitelline membrane” is a tri-layered membrane where L1 are suspended in a matrix. Stephanofilaria stilesi DNA was found in 5 out of the 13 cases in which adults or L1 were histologically observed (38%) and in 1 out of the 9 cases without adults or L1 present (11%). Phylogenetic analyses suggest a closer relationship of the genus Stephanofilaria with Thelazioidea, instead of the family Filariidae (Filarioidea), in which it has been historically allocated.ConclusionsOur study improved the characterization of lesions and described ultrastructural findings of S. stilesi and highlights that molecular tools should be utilized in combination with histology for improved diagnostic resolution.Graphical

Refugia or reservoir? Feral goats and their role in the maintenance and circulation of benzimidazole-resistant gastrointestinal nematodes on shared pastures.

Gastrointestinal nematodes threaten the productivity of grazing livestock and anthelmintic resistance has emerged globally. It is broadly understood that wild ruminants living in sympatry with livestock act as a positive source of refugia for anthelmintic-susceptible nematodes. However, they might also act as reservoirs of anthelmintic-resistant nematodes, contributing to the spread of anthelmintic resistance at a regional scale. Here, we sampled managed sheep and cattle together with feral goats within the same property in New South Wales, Australia. Internal transcribed spacer 2 (ITS-2) nemabiome metabarcoding identified 12 gastrointestinal nematodes (Cooperia oncophora, Cooperia punctata, Haemonchus contortus, Haemonchus placei, Nematodirus spathiger, Ostertagia ostertagi, Teladorsagia circumcincta, Oesophagostomum radiatum, Oesophagostomum venulosum, Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus rugatus). Isotype-1 β-tubulin metabarcoding targeting benzimidazole resistance polymorphisms identified 6 of these nematode species (C. oncophora, C. punctata, H. contortus, H. placei, O. ostertagi and T. circumcincta), with the remaining 3 genera unable to be identified to the species level (Nematodirus, Oesophagostomum, Trichostrongylus). Both ITS-2 and β-tubulin metabarcoding showed the presence of a cryptic species of T. circumcincta, known from domestic goats in France. Of the gastrointestinal nematodes detected via β-tubulin metabarcoding, H. contortus, T. circumcincta, Nematodirus and Trichostrongylus exhibited the presence of at least one resistance genotype. We found that generalist gastrointestinal nematodes in untreated feral goats had a similarly high frequency of the benzimidazole-resistant F200Y polymorphism as those nematodes in sheep and cattle. This suggests cross-transmission and maintenance of the resistant genotype within the wild ruminant population, affirming that wild ruminants should be considered potential reservoirs of anthelmintic resistance.

Trapping Behaviour of Duddingtonia flagrans against Gastrointestinal Nematodes of Cattle under Year-Round Grazing Conditions.

The purpose of using nematophagous fungi as biological control agents of gastrointestinal nematodes of livestock is to reduce the build-up of infective larvae on pasture and thus avoid clinical and subclinical disease. As the interaction of fungus-larval stages takes place in the environment, it is crucial to know how useful the fungal agents are throughout the seasons in areas where livestock graze all year-round. This study was designed to determine the predatory ability of the nematophagous fungus Duddingtonia flagrans against gastrointestinal nematodes of cattle during four experiments set up in different seasons. In each experiment, faeces containing eggs of gastrointestinal nematodes were mixed with 11,000 chlamydospores/g and deposited on pasture plots. A comparison between fungal-added faeces and control faeces without fungus were made with regard to pasture infectivity, larval presence in faecal pats, faecal cultures, faecal pat weight, and temperature inside the faecal mass. In three of the four experiments, Duddingtonia flagrans significantly reduced the population of infective larvae in cultures (68 to 97%), on herbage (80 to 100%), and inside the faecal pats (70 to 95%). The study demonstrated the possibility of counting on a biological control tool throughout most of the year in cattle regions with extensive grazing seasons.

Synergism of macrocyclic lactones against Haemonchus contortus.

A possible synergistic effect of macrocyclic lactones' (MLs) combination has been previously described against resistant gastrointestinal nematodes of cattle. In addition to synergism, drug-drug interactions between MLs can also result in additive or antagonistic effect, considering the different MLs pharmacokinetics, pharmacodynamics, and interactions with molecular mechanisms of resistance. Therefore, the aim of the current work was evaluated the effect of different MLs combinations against Haemonchus contortus. Infecting larvae of two isolates (one susceptible and one resistant to ivermectin) were used in the larval migration inhibition test. After estimating the half maximal effective concentration of abamectin (ABA), eprinomectin, (EPR), ivermectin (IVM), and moxidectin (MOX) for both isolates, combinations were delineated by a simplex-centroid mixture experiment, and the mixture regression analysis was applied to the special cubic model. A synergistic effect was found for the EPR + MOX against the susceptible isolate as well as the EPR + MOX, IVM + MOX, and ABA + EPR + IVM against the resistant isolate. An antagonistic effect of ABA + IVM + MOX was found against the susceptible isolate. For the susceptible isolate, a higher inhibition was found with greater proportions of EPR and lower proportions of the other drugs compared to the reference mixture. For the resistant isolate, inhibition greater than that of the reference mixture was found with higher proportions of IVM as well as lower proportions of the other drugs. The synergistic and antagonistic effects were dependent on the following: (a) parasite drug resistance profile, (b) the composition of the combination, and (c) the proportions used, with EPR and IVM exerting a greater impact on these effects.