Echinococcus granulosus ubiquitin-conjugating enzymes (E2D2 and E2N) promote the formation of liver fibrosis in TGFß1-induced LX-2 cells.

BACKGROUND: Cystic echinococcosis (CE) is a widespread zoonosis caused by the infection with Echinococcus granulosus sensu lato (E. granulosus s.l.). CE cysts mainly develop in the liver of intermediate hosts, characterized by the fibrotic tissue that separates host organ from parasite. However, precise mechanism underlying the formation of fibrotic tissue in CE remains unclear. METHODS: To investigate the potential impact of ubiquitin-conjugating enzymes on liver fibrosis formation in CE, two members of ubiquitin-conjugating (UBC) enzyme of Echinococcus granulosus (EgE2D2 and EgE2N) were recombinantly expressed in Escherichia coli and analyzed for bioinformatics, immunogenicity, localization, and enzyme activity. In addition, the secretory pathway and their effects on the formation of liver fibrosis were also explored. RESULTS: Both rEgE2D2 and rEgE2N possess intact UBC domains and active sites, exhibiting classical ubiquitin binding activity and strong immunoreactivity. Additionally, EgE2D2 and EgE2N were widely distributed in protoscoleces and germinal layer, with differences observed in their distribution in 25-day strobilated worms. Further, these two enzymes were secreted to the hydatid fluid and CE-infected sheep liver tissues via a non-classical secretory pathway. Notably, TGFß1-induced LX-2 cells exposed to rEgE2D2 and rEgE2N resulted in increasing expression of fibrosis-related genes, enhancing cell proliferation, and facilitating cell migration. CONCLUSIONS: Our findings suggest that EgE2D2 and EgE2N could secrete into the liver and may interact with hepatic stellate cells, thereby promoting the formation of liver fibrosis.

Seroprevalence and risk factors of Toxoplasma gondii in sheep and goats of North West Province, South Africa.

BACKGROUND: The protozoan parasite Toxoplasma gondii causes toxoplasmosis, one of the most prevalent parasitic zoonotic diseases with significant economic and public health implications worldwide. Infection with the parasite has a significant adverse effect on sheep and goat production and can frequently go undetected in the herd, resulting in abortions and weak or dead offspring. Although there are few studies on seroprevalence and risk factors associated with T. gondii infections in livestock in other provinces of South Africa, there is no data in the North West province. Therefore, a cross-sectional study was conducted to investigate the seroprevalence of T. gondii and risk factors associated with exposure in sheep and goats of the North West province of South Africa. Sera from 439 livestock (164 sheep and 285 goats) were collected and analysed for the presence of T. gondii IgG antibodies using indirect ELISA (Enzyme-linked immunosorbent assay). An assessment of potential risk factors in farms associated with seropositivity was also conducted using a structured questionnaire. RESULTS: Out of the 439 tested sheep and goats, 13.9% (61/439) were positive for IgG antibodies against T. gondii. Sheep and goats had seroprevalences of 19.5% (32/164) and 10.5% (29/275) respectively. In the multivariable logistic regression model, the risk of acquiring T. gondii was significantly higher in the mixed breed [Odds ratio (OR) = 71.07; 95% confidence interval (CI): 266.8-1893.1; p < 0.011)] animals than white dorper sheep and in farms that burn or bury aborted material (OR = 42.04; CI: 179.9-982.5; p = 0.020) compared to those that only burn aborted material. The risk was lower for the farms in Kagisano-Molopo (OR = 0.00; CI: 0.0-25.4; p = 0.015) and Mahikeng (OR = 0.00; CI: 0.0-4.9; p < 0.001) local municipalities than Greater Taung local municipality, and for the animals that drink water from dams (OR = 0.03; CI: 0.2-58.8; p = 0.021) than those that drink from boreholes. CONCLUSION: The seroprevalence and risk factors associated with transmission observed show that T. gondii infection is widespread in sheep and goats of the North West province.

Investigating hill sheep farmers and crofters' experiences of blackloss in the Highlands and Islands of Scotland.

Hill sheep farming is an important component of Scottish agriculture and comprises a significant land use in much of the Highlands and Islands. However it faces significant challenges due to the natural constraints of the landscape. Hill sheep farming uses hardy traditional breeds, such as the Scottish blackface and North Country Cheviot to graze extensive areas, where the sheep are not housed and tend to lamb on the open hill. Flocks are gathered several times a year for stock checks, husbandry, and health treatments. Between these handling events, stock will disappear and be unaccounted for. These unexplained losses are known as blackloss in the Highlands and Islands. Previously reported figures for annual lamb blackloss give an average of 18.6%. These losses are in addition to the known losses of lambs and represent a significant welfare and sustainability issue. High parasite burdens, predation, a photosensitisation disease known as plochteach or yellowses, and poor nutrition are often given as presumed reasons for blackloss. A questionnaire was developed to assess the experiences, impacts and understanding flock managers have of blackloss. Typology analysis using partitioning around medoids was used to cluster respondents into three distinct groups: 1- very large extensive farms and Sheep Stock Clubs, 2- medium sized farms, and 3- small-scale crofts. The responses of these groups were subsequently analysed to see if their experiences and perceptions of blackloss differed with relation to lamb health challenges and predation impacts. The groups reported similar health challenges, apart from Group 1 which had a significantly higher plochteach challenge. In terms of predators, Group 1 also perceived white-tailed eagles (Haliaeetus albicilla) as a much higher threat to their lambs than the other groups. It was observed that many of the respondents believed blackloss is inevitable and that predators pose a large threat to lambs. However, most agreed that reducing these losses is important and that understanding the causes would enable them to do so.

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep.

BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.

Revisiting anthelmintic resistance in sheep flocks from São Paulo State, Brazil.

Haemonchus contortus and Trichostrongylus colubriformis are the most important gastrointestinal nematodes causing serious losses in sheep production of tropical and subtropical regions. Prophylaxis of gastrointestinal nematode infections is based on anthelmintics use, but their frequent administration selects multiple-resistant parasites. To evaluate how the situation has changed over the last decades, the anthelmintic resistance status of gastrointestinal nematodes in sheep flocks was assessed in the current study and compared to previous surveys. In each one of the 15 flocks evaluated, animals (n ≥ 7) were allocated into at least five groups and treated as follows: 1) untreated control; 2) albendazole; 3) levamisole; 4) ivermectin; and 5) monepantel. If more animals were available, two additional groups were included: 6) closantel, and 7) moxidectin. The faecal egg count reduction test (FECRT) was carried out to evaluate the pre- and post-treatment using the SHINY tool. Haemonchus spp. was the most prevalent nematode from faecal cultures. The mean efficacy of albendazole was 40%. Only in two farms, levamisole presented a relatively high percentage of reduction in the FECRT about 90%, while ivermectin and moxidectin presented the worst mean efficacy of 34% and 21% among all farms, respectively. Like other anthelmintics, closantel demonstrated low efficacy (63%) across all farms evaluated. Monepantel presented an overall mean efficacy of 79%, but it was the only anthelmintic that presented efficacy ≥95%, in five farms. The results revealed that gastrointestinal nematodes with multiple anthelmintic resistance were prevalent in all 15 sheep herds. The research suggests that nematodes are becoming more and more resistant to various anthelmintic compounds, which has made the problem worse. This circumstance highlights the necessity to put into practice sustainable and long-lasting methods to prevent gastrointestinal nematode infections in sheep husbandry.

In-herd prevalence of Fasciola hepatica and Calicophoron / Paramphistomum spp. infections in German dairy cows with comparison of two coproscopical methods and establishment of real-time pyrosequencing for rumen fluke species differentiation.

Infections with liver and rumen flukes are among the most frequent parasitic diseases in cattle worldwide. In Europe, the predominant liver fluke species is Fasciola hepatica, and the recently rapidly spreading rumen flukes are mostly Calicophoron daubneyi and occasionally Paramphistomum leydeni. In this study, 1638 faecal samples from individual dairy cows from 24 northern and 18 southern German farms as well as one central German farm, all preselected for potential F. hepatica infection, were examined to determine in-herd prevalences of liver and rumen fluke infections. Furthermore, individual faecal egg counts (FECs) were determined in the northern and central German cows. On farms with patent F. hepatica infections, the mean in-herd prevalence was 15.8% in northern Germany, 41.6% in southern Germany and 14.0% in the central German farm. Rumen fluke infections resulted in high in-herd prevalences in all regions with a mean prevalence of 46.0% in northern, 48.4% in southern and 40.0% in central Germany. Individual FECs varied between 0.1 and 4.1 (mean 0.4) eggs per gram faeces (EPG) for F. hepatica and between 0.1 and 292.4 (mean 16.9) EPG for rumen flukes. Mean in-herd prevalence and mean FECs did not differ significantly between mono- and coinfected farms for either fluke species. Comparison of the classical sedimentation technique and the Flukefinder® method on a subset of 500 faecal samples revealed a similar number of positive samples, however, Flukefinder® mean FECs were three to four times higher for liver and rumen fluke eggs, respectively, with an increasing gap between EPG levels with rising egg counts. Fluke egg size measurement confirmed P. leydeni eggs on average to be larger in length and width (161.0 µm x 87.1 µm) than those of C. daubneyi (141.8 µm x 72.9 µm). However, due to overlap of measurements, morphological species identification based on egg size proved unreliable. For accurate identification, a real-time pyrosequencing approach was established, offering the advantage over classical Sanger sequencing of unambiguously identifying rumen fluke mixed species infections. Real-time pyrosequencing confirmed C. daubneyi (78.1% [50/64]) as the predominant rumen fluke species in Germany, while P. leydeni was detected in 12.5% (8/64) of sampled cows. A total of 9.4% (6/64) cows were infected with both C. daubneyi and P. leydeni, representing the first finding of a mixed infection in domestic ruminants in Europe to date.

Investigating the perceived versus actual gastrointestinal nematode challenge on extensive sheep farms.

Extensive farming systems form an integral part of sheep production systems across Europe. However, with innate production handicaps, declining sheep numbers and narrow economic margins, production is becoming increasingly challenging threatening the future sustainability of the industry. Gastrointestinal nematodes (GINs) are a significant cause of production losses to the global sheep industry, with well-established resistance to the major anthelmintic groups. Traditionally, extensive farming systems are not thought to have a significant parasite challenge compared with intensive farms, but there is a need to identify the scale and importance of GINs on extensive farms to inform the need for sustainable control strategies. In this study, a questionnaire of extensive farmers (n=34) was conducted and parasitological data were collected from nine study farms to investigate the perceived versus actual GIN and anthelmintic resistance challenge faced by extensive farms. The results showed a production-limiting challenge on most farms, with a higher GIN challenge observed on improved pastures. Furthermore, over half of the extensive farmers perceived anthelmintic resistance to be a greater problem for intensive farmers, with only 20% of respondents reporting known anthelmintic resistance. However, all study farms had evidence of resistance to at least one group of anthelmintics. Consequently, this study has demonstrated that despite the traditional perception of parasitism on extensive farms, there is a need to increasingly consider its impact and take a proactive approach to sustainable control, with solutions tailored to their unique management.

Efficacy of eprinomectin 5 mg/mL topical solution administered pour on at 1 mg per kg body weight against Oestrus ovis myiasis in sheep and goats.

The larvae of the nasal bot, Oestrus ovis, mainly parasitize sheep and goats and some species of wild Caprinae but other mammals and humans are also vulnerable to infestation. Eprinomectin 5 mg/mL topical solution (EPRINEX® Multi, Boehringer Ingelheim) administered at 1 mg eprinomectin per kg body weight pour on was recently authorized as an anthelmintic for sheep and goats with zero hours milk withdrawal in several countries in Europe. As the product in cattle has claims against a broad range of parasites including insect parasites and activity against O. ovis has previously been reported following extra-label use in sheep, its therapeutic efficacy against ovine and caprine O. ovis myiasis was evaluated in three regulatory compliant, masked clinical studies. Pre-study recovery of O. ovis larvae from five or six of six randomly selected animals per study site (Bulgaria, one site, sheep; Greece, two sites, sheep or goats) supported the inclusion of the animals from those sites into the studies. The study animals (34 animals per study) were ranked based on bodyweight and allocated randomly to remain untreated (control) or to be treated with eprinomectin 5 mg/mL topical solution at 1 mL per 5 kg body weight pour on. Treatment efficacy was determined based on O. ovis larval counts of eprinomectin 5 mg/mL topical solution-treated vs. untreated animals three weeks after treatment administration. Live O. ovis larvae, including all three instars in each study, were recovered from 13 or 16 of the 17 control animals in the sheep studies (range, 1 to 14 or 5 to 18 larvae, respectively) and from all 17 controls in the goat study (range, 7 to 18 larvae). In each study, eprinomectin 5 mg/mL topical solution-treated animals had significantly (p < 0.001) fewer live O. ovis larvae than the controls. Efficacy of the treatment was 100% and 91.3% against the combined parasitic O. ovis larval stages in sheep and in goats, respectively. The treatment was well accepted by all animals and no health problems were observed throughout the studies. The results of these studies demonstrated eprinomectin 5 mg/mL topical solution administered pour on at 1 mL per 5 kg body weight to be an efficacious and safe treatment of ovine and caprine oestrosis.

Application of the SeDeM system for the preparation of antiparasitic tablets from mesquite flour for use in sheep.

The use of herbal medicine to treat various diseases is becoming increasingly important as an alternative therapy. Numerous plants have been traditionally used for different purposes, including antiparasitic in humans and animals. Diseases caused by gastrointestinal parasites in ruminants, especially by the nematode Haemonchus contortus, cause large economic losses to the producers, whether by complications of the diseases or the cost of treatment. The main way of handling nematodiasis is by administering anthelmintic drugs, but their excessive use has the disadvantage of causing drug resistance; therefore, an alternative is the use of herbal medicine for this purpose. Mesquite (Prosopis spp.) has been used in Mexico to treat gastrointestinal diseases attributed to helminths. The present study aimed to characterize the rheological properties of mesquite flour using the SeDeM Expert System to determine its suitability for tablet production by direct compression. Direct compression technology facilitates the tableting process by reducing manufacturing costs. The results of the present study indicate that mesquite flour can be processed by direct compression. The latter could allow the manufacturing of economic tablets to treat infections by H. contortus in ruminants.

Genome-wide association studies of parasite resistance, productivity and immunology traits in Scottish Blackface sheep.

Gastrointestinal parasitism represents a global problem for grazing ruminants, which can be addressed sustainably by breeding animals to be more resistant against infection by parasites. The aim of this study was to assess the genetic architecture underlying traits associated with gastrointestinal parasite resistance, immunological profile and production in meat sheep, and identify and characterise candidate genes affecting these traits. Data on gastrointestinal parasite infection (faecal egg counts for Strongyles (FECS) and Nematodirus (FECN) and faecal oocyst counts for Coccidia, along with faecal soiling scores (DAG), characterised by the accumulation of faeces around the perineum) and production (live weight (LWT)) were gathered from a flock Scottish Blackface lambs at three and four months of age. Data on the immune profile were also collected from a subset of these lambs at two and five months of age. Immune traits included the production of Interferon-γ (IFN-γ), Interleukin (IL)-4 and IL-10 following stimulation of whole blood with pokeweed mitogen (PWM) or antigen from the gastric parasite Teladorsagia circumcincta (T-ci), and serum levels of T. circumcincta-specific immunoglobulin A (IgA). Animals were genotyped with genome-wide DNA arrays, and a total of 1 766 animals and 45 827 Single Nucleotide Polymorphisms (SNPs) were retained following quality control and imputation. Genome-wide association studies were performed for 24 traits. The effects of individual markers with significant effects were estimated, and the genotypic effect solutions were used to estimate additive and dominance effects, and the proportion of additive genetic variance attributed to each SNP locus. A total of 15 SNPs were associated at least at a suggestive level with FECS, FECN, DAG, IgA, PWM-induced IFN-γ and IL-4, and T-ci-induced IL-10. This study uncovered 52 genes closely related to immune function in proximity to these SNPs. A number of genes encoding C-type lectins and killer cell lectin-like family members were close to a SNP associated with FECN, while several genes encoding IL-1 cytokine family members were found to be associated with IgA. Potential candidate genes belonging to or in close proximity with the Major Histocompatibility Complex (MHC) were revealed, including Homeostatic Iron Regulator and butyrophilin coding genes associated with IFN-γ(PWM), and IL-17 coding genes associated with IgA. Due to the importance of the MHC in the control of immune responses, these genes may play an important role in resistance to parasitic infections. Our results reveal a largely complex and polygenic genetic profile of the studied traits in this Scottish Blackface sheep population.

Refugia, climatic conditions and farm management factors as drivers of adaptation in Nematodirus battus populations.

Parasites are known for their ability to rapidly adapt to changing conditions. For parasitic helminths, changes in climate, along with farming and management practices associated with the intensification of livestock farming, provide novel challenges which can impact on their epidemiology and control. The sustainability of livestock production partially relies on effective control of helminth infection. Therefore, understanding changes in parasite behaviour, and what drives these, is of great importance. Nematodirus battus is an economically important helminth in the UK and temperate regions. Its infective larvae typically overwinter in eggs on pasture and hatch synchronously in spring, causing acute disease in lambs. Attempts to control disease typically rely on whole-flock benzimidazole (BZ) treatments. In recent years, the emergence of BZ-resistance, alongside the hatching of eggs without the classical over-winter 'chill stimulus', have made N. battus more difficult to control. In three previous studies, after collecting a large number of N. battus populations alongside farm management data from commercial farms, we explored the prevalence of genetic mutations associated with BZ-resistance (n = 253 farms), the ability of eggs to hatch with and without a chill stimulus (n = 90 farms) and how farm management practices varied throughout the UK (n = 187 farms). In the present study, we identify factors which may be acting as drivers, or barriers, to either the development of resistance or the variable hatching behaviour of N. battus eggs. Generalised linear mixed effect models were applied to regress experimental hatching and genotyping data on farm management and additional environmental data. Both variable hatching and resistance development appeared associated with the maintenance of parasite refugia as well as grazing management, particularly reseeding of pasture routinely grazed by young lambs each spring and the practice of set-stocked grazing. Effective quarantine measures were identified as the main protective factor for the development of BZ-resistance whereas set stocked grazing and population bottlenecks, resulting from reseeding heavily contaminated pastures, were risk factors. Spring maximum temperature and other climatic factors were associated with 'typical' hatching of eggs following a chill stimulus whilst several management factors were linked with hatching without prior chilling. For example, practices which reduce parasite numbers on pasture (e.g. re-seeding) or restrict availability of hosts (e.g. resting fields), were found to increase the odds of non-chill hatching. Retention of the timing of lambing and infection level of the host within the fitted model indicated that requirement for a chill stimulus prior to hatching may be plastic, perhaps subject to change throughout the grazing season, in response to immune development or parasite density-dependence within the host. Further investigation of the influence of the factors retained within the fitted models, particularly the theme of parasite refugia which was highlighted in relation to both the presence of BZ-resistance alleles and alternative hatching, is required to establish robust, sustainable parasite control and farm management strategies.

The feeding of heather (Calluna vulgaris) to Teladorsagia circumcincta infected lambs reduces parasitism but can detrimentally impact performance.

Gastrointestinal nematode (GIN) infections impact small ruminant health, welfare, and production across farming systems. Rising anthelmintic resistance and regulation of synthetic drug use in organic farming is driving research and development of sustainable alternatives for GIN control. One alternative is the feeding of plants that contain secondary metabolites (PSMs) e.g., proanthocyanidins (PA, syn. condensed tannins) that have shown anthelmintic potential. However, PSMs can potentially impair performance, arising from reduced palatability and thus intake, digestibility or even toxicity effects. In this study, we tested the trade-off between the antiparasitic and anti-nutritional effects of heather consumption by lambs. The impact of additional feeding of a nematophagous fungus (Duddingtonia flagrans) on larval development was also explored. Lambs infected with Teladorsagia circumcincta or uninfected controls, were offered ad libitum heather, or a control chopped hay for 22 days during the infection patent period. Eight days into the patent period, parasitised lambs were supplemented (or remained unsupplemented) with D. flagrans for a 5-day period. Performance and infection metrics were recorded, and polyphenol levels in the heather and control hay were measured to investigate their association with activity. The lambs consumed heather at approximately 20% of their dry matter intake, which was sufficient to exhibit significant anthelmintic effects via a reduction in total egg output (P = 0.007), compared to hay-fed lambs; the magnitude of the reduction over time in heather fed lambs was almost 10-fold compared to control lambs. Negative effects on production were shown, as heather-fed lambs weighed 6% less than hay-fed lambs (P < 0.001), even though dry matter intake (DMI) of heather increased over time. D. flagrans supplementation lowered larval recovery in the faeces of infected lambs by 31.8% (P = 0.003), although no interactions between feeding heather and D. flagrans were observed (P = 0.337). There was no significant correlation between PA, or other polyphenol subgroups in the diet and egg output, which suggests that any association between heather feeding and anthelmintic effect is not simply and directly attributable to the measured polyphenols. The level of heather intake in this study showed no antagonistic effects on D. flagrans, demonstrating the methods can be used in combination, but provide no additive effect on overall anthelmintic efficacies. In conclusion, heather feeding can assist to reduce egg outputs in infected sheep, but at 20% of DMI negative effects on lamb performance can be expected which may outweigh any antiparasitic benefits.

Genetic profile of adaptive immune traits and relationships with parasite resistance and productivity in Scottish Blackface sheep.

Gastrointestinal (GI) parasites cause significant production losses in grazing ruminants which can be mitigated by breeding animals resistant to disease. Lymphocyte cytokine production and parasite-specific Immunoglobulin A (IgA) are adaptive immune traits associated with immunity to GI parasites. To explore the utility of these traits for selective breeding purposes, this study estimated the genetic parameters of the immune traits in sheep and assessed their relationship with disease and productivity traits. Whole blood stimulation assays were performed on 1 040 Scottish Blackface lambs at two months of age in 2016-2017. Blood was stimulated with either pokeweed mitogen (PWM), a non-specific activator of lymphocytes, and Teladorsagia circumcincta (T-ci) larval antigen to activate parasite-specific T lymphocytes. The type of adaptive immune response was determined by quantifying production of cytokines interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10, which relate to T-helper type (Th) 1, Th2 and regulatory T cell responses, respectively. Serum T-ci specific IgA was also quantified. Heritabilities were estimated for each immune trait by univariate analyses. Genetic and phenotypic correlations were estimated between different immune traits, and between immune traits vs. disease and productivity traits that were recorded at three months of age. Disease phenotypes were expressed as faecal egg counts (FEC) of nematode parasites (Strongyles and Nematodirus), faecal oocyst counts (FOC) of coccidian parasites, and faecal soiling score; production was measured as lamb live weight. Significant genetic variation was observed in all immune response traits. Heritabilities of cytokine production varied from low (0.14 ± 0.06) to very high (0.77 ± 0.09) and were always significantly greater than zero (P < 0.05). IgA heritability was found to be moderate (0.41 ± 0.09). Negative associations previously identified between IFN-γ production and FOC, and IL-4 production and strongyle FEC, were not evident in this study, potentially due to the time-lag between immune and parasitology measures. Instead, a positive genetic correlation was found between FOC and PWM-induced IFN-γ production, while a negative genetic correlation was found between FOC and T-ci induced IL-10. Live weight was negatively genetically correlated with IFN-γ responses. Overall, IFN-γ and IL-4 responses were positively correlated, providing little evidence of cross-regulation of Th1 and Th2 immunity within individual sheep. Furthermore, T-ci specific IgA was highly positively correlated with PWM-induced IL-10, indicating a possible role for this cytokine in IgA production. Our results suggest that while genetic selection for adaptive immune response traits is possible and may be beneficial for parasite control, selection of high IFN-γ responsiveness may negatively affect productivity.

Identification of sheep eimerian parasites, Eimeria crandallis and Eimeria faurei, employing microscopic and molecular tools.

Coccidiosis, caused by apicomplexan Eimeria species, is a protozoan disease that affects various species of wild and domestic animals. However, data available on Eimeria diversity in ruminants in Saudi Arabia is meagre. Therefore, this study was designed to investigate some eimerian parasites infecting sheep (Sawakni and Harrie breeds) using microscopy and molecular methods for the first time in Saudi Arabia. Twenty-four fecal samples were collected from sheep farms. Based on the floatation technique, eimerian oocysts were observed in 8 of the 24 (33.33%) fecal samples. The coccidian-positive samples were subjected to fecal culture in a shallow layer of 2.5% potassium dichromate (K2 Cr2 O7 ). Detected eimerian oocysts were described micromorphometrically as the basis for traditional oocyst identification. Morphologically, the sporulated oocysts were similar to those of sheep eimerian parasies; Eimeria faurei and Eimeria crandallis. PCR products from the two eimerian species detected from Sawakni and Harrie breeds were sequenced and were found to be distinct from each other with mutations at five positions. One of them clustered with E. crandallis with 99.8%-100% identity with sequences available in GenBank. E. crandallis was obtained from two Sawakni sheep and two Harrie sheep. The other sequences grouped with E. faurei with 99.8% identity with the only sequences available in GenBank. E. crandallis was detected from both Sawakni and Harrie breeds whereas E. faurei was detected only from Sawakni sheep. The findings of this study have implications for the importance of morphometric identification with advanced molecular tools to confirm the identities of sheep Eimeria species and to address the taxonomic study of this eimeriid parasite at the species level.

Rapid, automated quantification of Haemonchus contortus ova in sheep faecal samples.

Haemonchus contortus is one of the most pathogenic nematodes affecting small ruminants globally and is responsible for large economic losses in the sheep and goat industry. Anthelmintic resistance is rampant in this parasite and thus parasite control programs must account for drug efficacy on individual farms and, sometimes, whether H. contortus is the most prevalent trichostrongylid. Historically, coproculture has been the main way to determine the prevalence of H. contortus in faecal samples due to the inability to morphologically differentiate between trichostrongylid egg types, but this process requires a skilled technician and takes multiple days to complete. Fluoresceinated peanut agglutinin (PNA) has been shown to specifically bind H. contortus and thus differentiate eggs based on whether they fluoresce, but this method has not been widely adopted. The ParasightTM System (PS) fluorescently stains helminth eggs in order to identify and quantify them, and the H. contortus PNA staining method was therefore adapted to this platform using methodology requiring only 20 min to obtain results. In this study, 74 fecal samples were collected from sheep and analyzed for PNA-stained H. contortus, using both PS and manual fluorescence microscopy. The percentage of H. contortus was determined based on standard total strongylid counts with PS or brightfield microscopy. Additionally, 15 samples were processed for coproculture with larval identification, and analyzed with both manual and automated PNA methods. All methods were compared using the coefficient of determination (R2) and the Lin's concordance correlation coefficient (ρc). ParasightTM and manual PNA percent H. contortus results were highly correlated with R2 = 0.8436 and ρc = 0.9100 for all 74 fecal samples. Coproculture versus PS percent H. contortus were also highly correlated with R2 = 0.8245 and ρc = 0.8605. Overall, this system provides a rapid and convenient method for determining the percentage of H. contortus in sheep and goat fecal samples without requiring specialized training.

A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within ß-tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Seroprevalence and coprological prevalence of liver fluke Fasciola hepatica in cattle and sheep from Santander department, Colombia.

Fasciola hepatica is a parasite with a worldwide distribution that affects several mammals, including humans, and is considered a public health problem. Therefore, the aim of this study was to determine the prevalence of Fasciola hepatica in humans, cattle and sheep, as well as to evaluate factors associated with the prevalence. A total of 185 serum samples from sheep, 290 from cattle, and 114 from humans were collected and processed using an in-house developed ELISA to detect IgG antibodies against F. hepatica. Additionally, 185 stool samples from sheep and 290 from cattle were examined using a Dennis sedimentation technique. Risk factors were analyzed using epidemiological surveys. The overall seroprevalence was 46.5% (86/185) in sheep, 32.5% (94/289) in cattle, and no humans tested positive for the infection. The coprological prevalence was 47.7% (86/180) in sheep and 33.7% (98/290) in cattle. Female gender and cattle living with alternate grazing management showed 2.5 and 6.5 times higher probability of infection, respectively. Bovines coexisting with sheep exhibited a higher risk of infection (odds ratio [OR]=4.3) compared to those without sheep. We concluded that F. hepatica in cattle and sheep has an endemic behavior, and therefore represents a problem of public health for rural communities.

Molecular characterization of Cryptosporidium in ruminants and observation of natural infection by Cryptosporidium andersoni in sheep from Paraná, Brazil.

The aim of this study was to identify Cryptosporidium species found in cattle and sheep in Paraná, southern region of Brazil. Individual fecal samples from 458 bovines and 101 sheep were submitted for molecular analysis by PCR and nested PCR using specific primers for sequences of the 18S ribosomal unit (rRNA). Positive samples were analyzed using restriction fragment length polymorphism (RFLP), followed by genetic sequencing for species confirmation. The occurrence of Cryptosporidium was 11.27% (63/559). The highest occurrence was detected in lambs (12/59, 20.33%). From the 63 positive samples, it was possible to identify the species in 58 of them by RFLP and genetic sequencing. Five species of Cryptosporidium were identified: Cryptosporidium andersoni, Cryptosporidium bovis, Cryptosporidium ryanae, Cryptosporidium xiaoi, and Cryptosporidium parvum. The most prevalent species was C. andersoni (41.38%) and the least predominant was C. parvum (10.34%). The most abundant species of Cryptosporidium in dairy calves were C. andersoni (11/25) and C. ryanae (6/25). Of the 17 positive sheep, nine (52.94%) were infected with C. andersoni. This finding is the first report on the occurrence of C. andersoni in naturally infected sheep in Brazil and the first observation of a high absolute occurrence of this Cryptosporidium species in sheep.

Newly optimized ELISA kit and LAT reveal significantly higher seroprevalence in sheep raised in agro-ecological zone as against range-ecological zone, with a significant association of meteorological parameters.

BACKGROUND: Toxoplasma gondii is a zoonotic and foodborne intracellular parasite capable of inducing congenital infections, stillbirths and abortions in humans and animals, globally. The consumption of undercooked or raw mutton is "one of the vital risks" for acquiring toxoplasmosis: an asymptomatic condition in healthy persons, while life-threatening in immunodeficient individuals like "HIV/AIDS" patients. OBJECTIVES: The current study has multiple objectives: to optimize a newly ELISA kit for Sheep, to find out the seroprevalence of ovine toxoplasmosis of two ecological zones of the Punjab, Pakistan through LAT and newly Optimized Sheep ELISA kit, to do the comparison of efficacies of various tests (LAT with newly Optimized ELISA kit and newly Optimized ELISA kit with commercial ELISA kit) and to determine the different meteorological parameters as the risk factors for T. gondii infection in sheep. METHODS: A cross-sectional study was conducted on 400 sheep sera, 200 were collected from sheep raised on open grazing system by local farmers in the adjoining areas of Civil Veterinary Dispensaries (CVDs) of range-ecological zone i.e. tehsil Kot Chutta (Dera Ghazi khan). Similarly, the remaining 200 were collected from agro-ecological zone i.e. tehsil Sharaqpur (Sheikhupura), to evaluate the comparative efficacy of LAT with optimized ELISA kit and newly optimized ELISA kit with commercial ELISA kit. FINDINGS: The newly ELISA kit optimized against a commercial ELISA kit was found to have 100% sensitivity, 97.6% specificity with 98% Positive Predictive Value, 100% Negative Predictive Value, Cut off value = 0.505, 28.28 LR+, 0.0104 LR-, and 2719.23 DOR. Seroprevalence of toxoplasmosis was detected significantly (P < 0.01; χ2) higher in Sharaqpur (44.5% by LAT; 35.5% by ELISA) as compared to that in Kot Chutta (39.5% by LAT; 31% by ELISA). The highest seroprevalence was seen in the sheep of the 1-2 years age group (P < 0.01; χ2), whereas the lowest in the oldest animals (≥ 4 years). Investigation of meteorological data of both the regions reveals that the zone with higher seroprevalence has relatively higher rainfall, higher humidity, lower environmental temperatures, and higher altitude as the critical factors, potentially behind the significant difference seen in seroprevalence level. The partial correlation of both tests (newly optimized ELISA kit and LAT) was 0.991 at maximum temperature in Sharaqpur while it was 0.981 in Kot Chutta. INTERPRETATION: A novel significant correlation was found between the meteorological parameters (relative humidity, minimum, maximum, and average temperatures) divided into yearly units of both the ecological zones, and year-wise seroprevalence (birth years of age-wise groups) of the corresponding regions. We hypothesize that such environmental conditions increase the risk of toxoplasmosis in grazing sheep, owing to a more favorable environment for coccidian oocyst survival. The ELISA kit optimized in this study will be helpful for the detection of seroprevalence of ovine toxoplasmosis in other ecological zones of Pakistan as well as of any other country in the world. More studies are recommended involving regions from other ecological zones of Pakistan to further explore the seroprevalence of ovine toxoplasmosis and to ratify the novel correlation of meteorological parameters with seroprevalence.

Climate change effects on terrestrial parasitic nematodes: Where are the knowledge gaps?

Climate change is expected to affect parasitic nematodes and hence possibly parasite-host dynamics and may have far-reaching consequences for animal health, livestock production, and ecosystem functioning. However, there has been no recent overview of current knowledge to identify how studies could contribute to a better understanding of terrestrial parasitic nematodes under changing climates. Here we screened almost 1,400 papers to review 57 experimental studies on the effects of temperature and moisture on hatching, development, survival, and behaviour of the free-living stages of terrestrial parasitic nematodes with a direct life cycle in birds and terrestrial mammals. Two major knowledge gaps are apparent. First, research should study the temperature dependency curves for hatching, development, and survival under various moisture treatments to test the interactive effect of temperature and moisture. Second, we specifically advocate for more studies that investigate how temperature, and its interaction with moisture, affect both vertical and horizontal movement of parasitic nematodes to understand infection risks. Overall, we advocate for more field experiments that test environmental effects on life-history traits and behaviour of parasitic nematodes in their free-living stages under natural and realistic circumstances. We also encourage studies to expand the range of used hosts and parasitic nematodes because 66% of results described in the available studies use sheep and cattle as hosts and 32% involve just three nematode species. This new comprehension brings attention to understudied abiotic impacts on terrestrial parasitic nematodes and will have broader implications for livestock management, wildlife conservation, and ecosystem functioning in a rapidly warming climate.