Preliminary results of the recombinase polymerase amplification technique for the detection of Haemonchus contortus from Hungarian field samples.

Haemonchus contortus is a parasitic nematode of small ruminants responsible for significant economic losses and animal health concerns globally. Detection of gastrointestinal nematode (GIN) infection in veterinary practice typically relies on microscopy-based methods such as the faecal egg count and morphological identification of larval culture. However, mixed co-infections are common and species-specific identification is typically time-consuming and expertise-intensive. Compounded by increasing anthelmintic resistance, there is an urgent need to implement the molecular diagnosis of GIN in the livestock industry, preferably in field settings. Advances in isothermal amplification techniques including recombinase polymerase amplification (RPA) assays could improve this. Yet, constraints in RPA kit availability and amplicon detection systems limit the use of this technology in point of care settings. In this study, we present an early-stage, proof-of-concept demonstration of RPA targeting the internal transcribed spacer (ITS2) region of H. contortus. Having tested against eight closely related nematodes and also against five farm isolates in Eastern Hungary, preliminary results derived from a comparative analysis of 3 primer sets showed the assay detects H. contortus DNA and has a limit of detection of 10-5 ng/µl. We also tested an end-result naked eye detection system using various DNA binding dyes, of which EvaGreen® dye was successful for a qualitative RPA detection that could be adaptable at farm sites.

Portable near-infrared spectroscopy: A rapid and accurate blood test for diagnosis of Haemonchus contortus infection and for targeted selective treatment of sheep.

Haemonchus contortus is the most prevalent and important gastrointestinal nematode (GIN) in small ruminants. Since it reduces the packed cell volume (PCV), causing anemia, early diagnosis can be used for targeted selective treatment (TST) of sheep, reducing antiparasitic drug use and anthelmintic resistance. This study aimed to predict PCV values through near-infrared spectroscopy (NIRS) and to develop a classification and diagnosis model of H. contortus infection using PCV values, eggs per gram of feces (EPG) counts and mean daily weight gain (DWG). A total of 1728 spectra were collected from blood samples of 216 lambs with a portable NIR spectroscope. In parallel, other parameters indicative of infection were measured: PCV by hematocrit, FAffa MAlan CHArt (FAMACHA) scores, EPG and DWG. To evaluate the relationship between NIRS spectra and the evaluated parameters, principal component analysis (PCA) was used for an exploratory analysis, regression by the partial least squares method (PLS) for the prediction of PCV values via NIRS, and PCA linear discriminant analysis (PCA-LDA) as a classification model for diagnosis. The absorption peaks in the NIRS region associated with the excitation of overtones of nitrogen-hydrogen (N-H) functional groups of proteins had a strong impact on the principal components (PCs), indicating that blood proteins, especially hemoglobin, can be estimated by the NIRS technique. The model for predicting PCV by PLS presented a standard error of prediction of 2.53, root-mean-square error of 2.48, and coefficient of determination of 0.84, indicating good correlation between the PCV values predicted by the model and the PCV obtained by hematocrit. The PCA-LDA model presented 93.33% sensitivity and 82.18% accuracy, both higher than those of the FAMACHA method, as was expected for resilient Morada Nova lambs. The multivariate models associated with the NIRS technique reported here can be used in the future as a quick and versatile tool for H. contortus infection diagnosis and TST application in lambs.

Point of care colourimetric and lateral flow LAMP assay for the detection of Haemonchus contortus in ruminant faecal samples.

In this study, we present an optimised colourimetric and a lateral flow LAMP assay for the detection of Haemonchus contortus in small ruminant faecal samples. Using a previously published LAMP primer set, we made use of commercially available colourimetric LAMP and lateral flow kits and combined this into an optimised diagnostic assay which was then tested on field faecal samples from Eastern and South-Eastern Hungary as well as a pure H. contortus egg faecal sample from Kosice, Slovakia. Both assays showed no conflicts in visual detection of the results. Additionally, we modified and tested several centrifuge-free DNA extraction methods and one bead-beating egg lysis DNA extraction method to develop a true point of care protocol, as the source of the starting DNA is the main rate-limiting step in farm-level molecular diagnosis. Out of the various methods trialed, promising results were obtained with the magnetic bead extraction method. Sample solutions from the Fill-FLOTAC® technique were also utilised, which demonstrated that it could be efficiently adapted for field-level egg concentration to extract DNA. This proof of concept study showed that isothermal amplification technologies with a colourimetric detection or when combined with a lateral flow assay could be an important step for a true point of care molecular diagnostic assay for H. contortus.

TITLE: Dosage LAMP colorimétrique et à flux latéral pour la détection au point d'intervention d'Haemonchus contortus dans les échantillons de selles de ruminants. ABSTRACT: Dans cette étude, nous présentons un test colorimétrique optimisé et un test LAMP à flux latéral pour la détection d'Haemonchus contortus dans des échantillons de selles de petits ruminants. À l'aide d'un ensemble d'amorces LAMP publié précédemment, nous avons utilisé des kits colorimétriques LAMP à flux latéral disponibles dans le commerce et les avons combinés dans un test de diagnostic optimisé qui a ensuite été testé sur des échantillons de matières fécales de terrain provenant de l'est et du sud-est de la Hongrie ainsi que d'un échantillon d'œufs de H. contortus provenant de selles de Kosice, Slovaquie. Les deux tests n'ont montré aucun conflit dans la détection visuelle des résultats. De plus, nous avons modifié et testé plusieurs méthodes d'extraction d'ADN sans centrifugation et une méthode d'extraction de l'ADN des œufs par lyse par billes pour développer un véritable protocole de point d'intervention, car la source d'ADN de départ est la principale étape limitante du diagnostic moléculaire au niveau de la ferme. Parmi les différentes méthodes testées, des résultats prometteurs ont été obtenus avec la méthode d'extraction par billes magnétiques. Des solutions d'échantillons de la technique Fill-FLOTAC® ont également été utilisées, ce qui a démontré qu'elle pouvait être efficacement adaptée à la concentration d'œufs sur le terrain pour extraire l'ADN. Cette étude de preuve de concept a montré que les technologies d'amplification isotherme avec une détection colorimétrique ou lorsqu'elles sont combinées avec un test de flux latéral pourraient être une étape importante pour un véritable test de diagnostic moléculaire au point d'intervention pour H. contortus.

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Epidemiology and spatio-temporal distribution of gastrointestinal parasites infection and accuracy of FAMACHA test in sheep in traditional farming systems in Burkina Faso.

Blood and faecal samples from 2086 Sahelian and Djallonke sheep have been collected to assess the epidemiology and spatio-temporal distribution of gastrointestinal parasites. A survey with a questionnaire allowed collecting data on individual animal and farms. Faecal samples were analysed using the McMaster technique, while anaemia was assessed using FAMACHA score test and packed cell volume. The overall prevalence of gastrointestinal parasites was 91%, and strongyles (70.7%), Eimeria sp. (60.4%), Moniezia sp. (5.7%), Stronyloides sp. (0.9%) were identified. There was no significant association between the region and strongyles infection (P ˃ 0.05). Animals sampled during the rainy season (n = 565; P = 82.2%, mean EPG = 154.8 ± 12.02) were mostly and heavily infected than those from the hot dry (n = 518; P = 74.2%; mean EPG = 97.7 ± 17.4) and the cold dry season (n = 392; P = 55.5%; mean EPG = 24.5 ± 19.5) (P ˂ 0.05). Females (n = 1027; P = 72.1%; mean EPG = 77.6 ± 17) were most infected than males (n = 448; P = 67.7%; mean EPG = 61.6 ± 19.5) (P ˂ 0.05). Animals that were treated, at most 3 months before sampling (n = 166; P = 61.5%; mean EPG = 41.7 ± 21.4), were less infected than those, which received this treatment after three months before sampling (n = 199; P = 74.8%; mean EPG = 89.1 ± 15.8) (P ˂ 0.05). Data from blood samples indicate that 6.5% of sheep was anaemic (PCV ≤ 19%), while 59.7% was noted anaemic regarding FAMACHA scores. PCV values varied significantly for FAMACHA scores, and the highest mean value was noted for score 1 (27.6) (P ˂ 0.05). The Cohen's kappa score between FAMACHA and PCV was 0.02. These findings suggest that strongyles infections are significantly associated with season, and heavier infections were noted during rainy season.

Relationships between FAMACHA© scores and parasite incidence in sheep and goats.

One of the most important activities in animal raising is parasite control. Since parasites share what animals eat on one hand, they have disease-causing effects on the other hand, they are parasites that must be fought. In fact, they can be responsible for the decrease in yield and can also cause death. Haemonchus contortus parasite from the Trichostrongylidae family is a blood parasite that causes anemia in sheep and goats. In this study, using the FAMACHA© card, which is widely used in the determination of anemia caused by Haemonchus contortus in the world, the color of the conjunctiva was scored in Akkaraman sheep and Hair goats raised in the Sizma district of Konya, and the relations between the score and the parasite type and density were determined. Accordingly, while no animals with 1 and 5 scores were observed in sheep, the numbers of animals with scores of 2, 3, and 4 were determined as 7.84%, 40.2%, and 52.96%, respectively. Parasite eggs belonging to the Trichostrongylidae family were observed in 96.07% of the sheep. On the other hand, no animal with score 1 or 2 was observed in goats. The numbers of animals with 3, 4, and 5 scores were determined as 6.12%, 54.08%, and 39.80%, respectively. Eimeria, the most common parasite species, was found in 68.18% of the goats and parasite eggs from the Trichostrongylidae family were observed in 17.05%. As a result, it was concluded that the card in question was a practical, easily available, and cheap tool in the detection of anemia by using the relationships between FAMACHA© card scoring and parasite loads.

Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods.

This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

Development of a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay for the Detection of Haemonchus contortus in Goat Feces.

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.

Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods / Resistência anti-helmíntica de Haemonchus contortus em rebanhos ovinos no Brasil: concordância de métodos in vivo e in vitro (RESISTA-Test©)

Abstract This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

Resumo Este estudo avaliou o status de resistência de Haemonchus contortus em rebanhos ovinos do estado de São Paulo, Brasil, através de um estudo comparativo entre o teste de redução da contagem de ovos nas fezes (TRCOF) e o teste de desenvolvimento larvar (TDL). Para o TRCOF, 35 ovinos foram selecionados em cada um dos cinco rebanhos e divididos em grupos tratados com: benzimidazol, levamisol, ivermectina, monepantel e controle. Fezes foram coletadas para OPG e coproculturas. O TDL foi realizado com tiabendazol-TBZ, levamisol-LEV, ivermectina aglicona-IVM-A e Zolvix-ZLV. Resistência a todos os fármacos foi detectada por meio do TRCOF em 100% dos rebanhos, exceto para ZLV (40% resistentes e 20% suspeitos de resistência). O LDT indicou resistência ao TBZ e IVM-A em todos os rebanhos; em 80% dos rebanhos ao LEV e 10% ao ZLV. Foi obtida concordância total entre os dois testes para TBZ e IVM (k = 1,0), enquanto para LEV (k = 0,8) e ZLV (k = 0,9) foram obtidas concordância substancial e quase perfeita, respectivamente. A concordância entre os testes foi significante, sendo possível usar o resultado do LDT para prever o do TRCOF, validando-se o primeiro como um teste diagnóstico rápido para criadores de ovinos no Brasil.

Ovine haemonchosis: a review.

Sheep farming is the backbone of a rural economy in developing countries, and haemonchosis is a major impediment in the way of its progress. Haemonchus contortus (H. contortus) infection persists all over the world particularly in the tropical and sub-tropical regions. Various review articles have been published to substantially cover one or more aspects of its morphology, prevalence, pathogenesis, symptoms, diagnosis, immune response, drug resistance, treatment, and control measure. The objective of this paper is to briefly review past and present information available in the aforementioned areas in one place to enable the readers to fully understand the problem from a broader perspective. H. contortus parasite harbours in abomasum of affected animal and feeds on its blood, producing mild to severe symptoms and even death in acute form. The parasite thus inflicts heavy production losses and is of economic importance. H. contortus has developed diverse characters over the years leading to limited success in the production of vaccines. Indiscriminate use of the anthelmintics has produced drug resistance against almost all conventional products. Efficacy of medicinal plants and non-conventional chemicals has been reported under controlled experiments; however, research on their adverse effects on growth and fertility is yet to be studied. Research on molecular tools for identification and introduction of resistant genes into the flock is also underway but still a long journey to find its field application. Crossbreeding may compromise the production traits of the existing flock. In given circumstances, a targeted selective treatment approach along with selective breeding, culling of more susceptible animals, and maintaining a good body condition score through the provision of a balanced diet remains a workable strategy to control haemonchosis in sheep.

Influence of environmental factors on the detection of blood in sheep faeces using visible-near-infrared spectroscopy as a measure of Haemonchus contortus infection.

BACKGROUND: Existing diagnostic methods for the parasitic gastrointestinal nematode, Haemonchus contortus, are time consuming and require specialised expertise, limiting their utility in the field. A practical, on-farm diagnostic tool could facilitate timely treatment decisions, thereby preventing losses in production and flock welfare. We previously demonstrated the ability of visible-near-infrared (Vis-NIR) spectroscopy to detect and quantify blood in sheep faeces with high accuracy. Here we report our investigation of whether variation in sheep type and environment affect the prediction accuracy of Vis-NIR spectroscopy in quantifying blood in faeces. METHODS: Visible-NIR spectra were obtained from worm-free sheep faeces collected from different environments and sheep types in South Australia (SA) and New South Wales, Australia and spiked with various sheep blood concentrations. Spectra were analysed using principal component analysis (PCA), and calibration models were built around the haemoglobin (Hb) wavelength region (387-609 nm) using partial least squares regression. Models were used to predict Hb concentrations in spiked faeces from SA and naturally infected sheep faeces from Queensland (QLD). Samples from QLD were quantified using Hemastix® test strip and FAMACHA© diagnostic test scores. RESULTS: Principal component analysis showed that location, class of sheep and pooled versus individual samples were factors affecting the Hb predictions. The models successfully differentiated 'healthy' SA samples from those requiring anthelmintic treatment with moderate to good prediction accuracy (sensitivity 57-94%, specificity 44-79%). The models were not predictive for blood in the naturally infected QLD samples, which may be due in part to variability of faecal background and blood chemistry between samples, or the difference in validation methods used for blood quantification. PCA of the QLD samples, however, identified a difference between samples containing high and low quantities of blood. CONCLUSION: This study demonstrates the potential of Vis-NIR spectroscopy for estimating blood concentration in faeces from various types of sheep and environmental backgrounds. However, the calibration models developed here did not capture sufficient environmental variation to accurately predict Hb in faeces collected from environments different to those used in the calibration model. Consequently, it will be necessary to establish models that incorporate samples that are more representative of areas where H. contortus is endemic.

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat.

BACKGROUND: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. RESULTS: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 µg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. CONCLUSION: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Development and field evaluation of a species-specific mt-COI targeted SYBR-Green Real Time PCR for detection and quantification of Haemonchus contortus in cattle in Turkey.

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per µl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite.

Immunodiagnostic potential of recombinant tropomyosin during prepatent Haemonchus contortus infection in goat.

Excretory and secretory products (ESPs) are released by the parasites during Haemonchus contortus (H. contortus) infection. In this study, Tropomyosin (TpMy), one of these ESPs was used to develop western blotting and optimized Enzyme Linked immunosorbent assay (ELISA) for detection of H. contortus during early infection in goat. Microscopic examination was performed parallel for comparison. Recombinant tropomyosin protein was purified successfully. Western blotting results revealed that anti-recombinant H. contortus Tropomyosin (rHc-TpMy) antibodies could recognize the natural proteinand rHc-TpMy antigen did not show any cross-reaction with goat anti-sera of Fasciola hepatica, Trichinella spiralis, and Toxoplasma gondii. Moreover, initial antibodies were detected by both western blotting and indirect ELISA at 14 days post infection (DPI) and persisted till 30 DPI but fecal eggs count couldn't detect the eggs in feces at early stage (7 and 14 DPI). The optimized antigen coating concentration was calculated as 10 µg/ml (P/N Optimum Density450 = 4.165) with optimized dilution of serum (1:50) and secondary antibody (1:2500). Positive and negative cutoff value of the indirect-ELISA assay was calculated as 0.392 and 0.344, respectively. Receiver operating characteristic curve analysis validated the cutoff value (0.392) based on a high specificity and sensitivity. Indirect ELISA showed 90% diagnostic sensitivity and 100% diagnostic specificity. In comparison of serological and conventional method, rHc-TpMy based indirect ELISA showed more positive results (30%; 9/30) than microscopic examination (20%; 6/30). These results demonstrated that rHc-TpMy is a potential immunodiagnostic antigen to detect specific antibodies at early stage of infection in goat and serological methods are more reliable as compared to microscopic examination.

Sensitivity and specificity of the FAMACHA© system in tropical hair sheep.

The aim of this research was to evaluate the sensitivity and specificity of the FAMACHA© (F©) system in Morada Nova ewes. The conjunctivae of 562 ewes were evaluated using the F© system. Packed cell volume (PCV) served as the gold standard for clinical F© evaluation. To calculate the sensitivity and specificity of the F© system, different criteria were adopted: animals classified as (I) F© 4 and 5 or (II) 3, 4, and 5 were considered to be anemic and animals classified as (I) F© 1, 2, and 3, or (II) 1 and 2 were considered to be non-anemic. Three standard values of PCV, namely, ≤ 19%, ≤ 18%, or ≤ 15%, were used to confirm anemia. The percentage of correct treatments was always high when the F© values 4 and 5 were used as criteria for positive tests. For all the PCV cut-off values, more animals were classified as false positives when evaluated using F© 3, 4, and 5 as criteria for a positive test and more true negative animals when evaluated using only F© 4 and 5 as criteria for a positive test. For both sets of criteria for the positive tests, few animals were classified as false negatives and true positives. Eliminating the classification of F© 3 as anemic decreased the sensitivity and increased the specificity for all the PCV cut-off values for the ewes. The F© system can be used as a reliable alternative to reduce selection pressure for anthelmintics in relation to routine non-selective blanket treatment for worm control in the Morada Nova ewes.

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples.

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

Supplementation with rumen-protected proteins induces resistance to Haemonchus contortus in goats.

Resistance to gastro-intestinal nematode (GIN) in small ruminant is expected to arise from protein-rich rather than from energy-rich feeds. The objective of this study was to investigate the effect of the quality of the dietary proteins on the response of Creole goats to Haemonchus contortus. Three diets were compared: no supplementation (Hay: hay ad libitum), Control supplement (CS: hay ad libitum +2% BW of CS at 70 g of by-pass proteins/kg) and supplement enriched in rumen-protected proteins (RPP: hay ad libitum +2% BW of RPP at 139 g of by-pass proteins/kg). The FEC (faecal eggs counts) and the TFEC (total faecal eggs excreted/day) were significantly lower in the RPP. No difference was found between the supplemented diets for the total number of nematodes, but the RPP reduced the parasite prolificacy. The highest IgA responses were observed in animals with the highest nematode burden (Hay compared with CS diets). However, while the FEC and the TFEC were lower in animals feed with the RPP the IgA response were similar to those of the Hay. The IgA response that control GIN egg production in sheep could be one mediator of the resistance to H. contortus induced with by-pass proteins in goats.

Is the Famacha© system always the best criterion for targeted selective treatment for the control of haemonchosis in growing lambs?

Targeted selective treatment (TST) aims to aid in the control of gastrointestinal nematodes in small ruminants, identifying animals that require treatment with the purpose of preserving the Refugia population and delaying the buildup of anthelmintic (AH) resistance. The objective of this study was to evaluate the effectiveness of both productive and clinical indicators in the selective treatment of gastrointestinal parasites in growing lambs. Two experiments were carried out between 2015 and 2016. Experiment 1: lambs (n = 70) exclusively raised on pasture were evaluated from the ages of 60 days to 180 days old. Experiment 2: lambs (n = 48) raised on pasture and fed concentrated commercial supplementation were evaluated from the ages of 60 days to 150 days old. Parasitological (eggs per gram of feces - EPG), productive (average daily weight gain - DWG), and clinical (hematocrit - Ht and FAMACHA© system - F) parameters were measured every 14 days. The lambs were divided into 4 experimental groups with different criteria for AH treatment: the control group (CG), was treated with AH every 30 days; the FAMACHA© group (FG), in which lambs classified as F3, F4, and F5 were treated with AH; Daily weight gain group (DWGG), in which lambs with average DWG ≤ the average DWG of the CG minus one standard deviation were treated with AH; FAMACHA© + daily weight gain group (FDWGG), in which lambs classified as F3, F4 and F5 and/or lambs with average DWG ≤ the average DWG of the CG minus one standard deviation were treated with AH. In experiment 1, no significant differences in the general means of DWG and Ht (p > 0.05) were observed between experimental groups, although the lowest mean EPG was observed in the CG (p < 0.05). In experiment 2, the lowest mean EPG (p < 0.05) was also observed in the CG (1044.5 EPG), and the highest mean (p < 0.05) in the FG (4845.5 EPG). There were differences in the mean DWG of the CG (198.4 g) and FDWGG (200.0 g), which were both higher (p < 0.05) than that of the FG (90.5 g), and similar (p > 0.05) to the DWGG (151.8 g). In both experiments, considering the total mean number of AH treatments, FG had the lowest number of lambs treated with AH (p < 0.05), with the DWG and FDWGG being similar to each other (p > 0.05). Even when Haemonchus is the major parasite, it is not recommended to use the FAMACHA© system as an exclusive criterion for TST in growing lambs. The DWG productive criterion can be used effectively in the TST of growing lambs.

Droplet digital polymerase chain reaction (ddPCR) as a novel method for absolute quantification of major gastrointestinal nematodes in sheep.

In this paper, we present for the first time a new tool, based on Droplet Digital™ Polymerase Chain Reaction (ddPCR), for absolute quantification of key genera of gastrointestinal (GI) nematode parasites of grazing livestock. Four combinations of primers/probe sets targeting the internal transcribed spacer region 2 (ITS2) of the ribosomal RNA gene array were designed using the Primer3 software, following in silico analysis of nucleotide sequences from nematodes of interest downloaded from common databases. The amplified regions include both a universal region for detection of any strongylid gastrointestinal parasite and three different genus specific regions, making it possible to differentiate between the most important GI nematodes of sheep in Sweden: Haemonchus, Teladorsagia and Trichostrongylus. Analysis of samples containing serial dilutions and different mixtures of genomic DNA extracted from different species of adult worms proved useful in assessment of different threshold settings with the QuantaSoft software. Analysis of template DNA from these worms indicated that ddPCR is a viable choice for detection and absolute quantification of the different genera and also in samples with multiple species. Interpretation of the ddPCR results was straightforward and choice of analytical approach had little influence on the final results. Thus, the results obtained in the different analytical approaches seemed to be robust and the concentrations determined were uniform. Furthermore, the linear range of the Haemonchus ddPCR assay was similar to that of real-time PCR (qPCR). Taken together, our data confirm the suitability of ddPCR for detection and absolute quantification of three major sheep pathogens when tested on larval cultures from pooled ovine faeces. The results also indicate that ddPCR can be a useful complement to applications based on conventional egg counting methods such as the faecal egg reduction test (FECRT).

Helminth infections and hybridization between Haemonchus contortus and Haemonchus placei in sheep from Santana do Livramento, Brazil / Infecções helmínticas e hibridização entre Haemonchus contortus e Haemonchus placei em ovinos criados em Santana do Livramento, Brasil

Abstract The occurrence and intensity of helminth infections were evaluated in sheep from pastures shared with cattle. In 2015 and 2016, young male sheep acquired in Santana do Livramento, Rio Grande do Sul, Brazil, were finished in integrated crop-livestock system. We selected the 12 sheep that showed the highest number of nematode eggs per gram of faeces to search for worms in the gastrointestinal tract. Haemonchus contortus and Trichostrongylus colubriformis were the major parasites. H. contortus presented mean intensities of 1,159 and 257 worms in 2015 and 2016, respectively. T. colubriformis displayed mean intensities of 4,149 and 2,427 worms in 2015 and 2016, respectively. Of the 127 male specimens of Haemonchus spp. analysed by Polymerase Chain Reaction (PCR), 125 were H. contortus, one Haemonchus placei and one hybrid. Other species detected were Cooperia punctata , Cooperia pectinata, Cooperia spatulata, Cooperia curticei, Ostertagia ostertagi, Teladorsagia circumcincta , Trichostrongylus axei, Nematodirus spathiger , and Trichuris ovis. Twenty lambs presented cysts of Taenia hydatigena in the liver and mesentery. One lamb presented Coenurus cerebralis, the larval stage of Taenia multiceps, in the brain. In conclusion, sheep from pasture shared with cattle presented a high diversity of nematode species. H. contortus and H. placei co-infection occur with consequent hybridization.

Resumo A ocorrência de infecções helmínticas foi avaliada em ovinos que compartilhavam pastagem com bovinos. Em 2015 e em 2016, cordeiros machos foram adquiridos em Santana do Livramento, Rio Grande do Sul, para serem terminados em sistema de lavoura - pecuária (ILP). Em cada ano, 12 cordeiros que tinham maior contagem de ovos nas fezes foram abatidos para recuperação dos vermes. Haemonchus contortus e Trichostrongylus colubriformis foram as principais espécies registradas. H. contortus apresentou intensidade média de 1159 e 257 vermes em 2015 e 2016, respectivamente. T. colubriformis apresentou intensidade média de 4149 e 2427 parasitas em 2015 e 2016, respectivamente. De 127 machos de Haemonchus spp. analisados por "Polymerase Chain Reaction" (PCR), 125 foram identificados como H. contortus, um como Haemonchus placei e um como híbrido. Cooperia punctata, Cooperia pectinata, Cooperia spatulata, Cooperia curticei, Ostertagia ostertagi, Teladorsagia circumcincta , Trichostrongylus axei, Nematodirus spathiger e Trichuris ovis foram as outras espécies de nematódeos registradas. Vinte cordeiros apresentaram cistos de Taenia hydatigena no mesentério e no fígado. Um cordeiro apresentou no cérebro Coenurus cerebralis, o estágio larval de Taenia multiceps . Em conclusão, ovinos criados com bovinos apresentam grande diversidade de nematódeos. A co-infecção de H. contortus e H. placei favorece a produção de híbridos.