Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

The objectives of the present study were: (a) to describe the prevalence of helminth infections from pooled faecal samples from goat herds across Greece, (b) to evaluate herd-related factors potentially associated with the presence of these infections in the herds and (c) to compare the findings to those of a previous relevant study performed in sheep flocks. A cross-sectional study was carried out in 119 goat herds around Greece. Helminths were recovered from samples from 93.3% of herds: Dicrocoelium dendriticum (from 15.1% of herds), Paramphistomum cervi (from 0.8% of herds), Moniezia spp. (from 25.2% of herds), Trichostrongylidae (from 89.1% of herds), Nematodirus spp. (from 16.8% of herds), Strongyloides papillosus (from 5.0% of herds), Trichuris spp. (from 18.5% of herds) and lungworms (from 23.5% of herds). Mean epg counts in all herds in the study were 219 epg. In multivariable analyses, for \'high (> 300) epg counts in faecal samples\' the age of kid removal from their dams (p = 0.045) was found to be a significant factor, for \'high proportion (> 64%) of Teladorsagia spp. in faecal samples\' the month of the start of the kidding season (p = 0.045) was a significant factor, for \'high proportion (> 27%) of Haemonchus contortus in faecal samples\' nutritional modifications during gestation (p = 0.002) and application of reproductive control practices in the farm (p = 0.013) were the significant factors and for \'presence of D. dendriticum in faecal samples\' the number of veterinary visits to the farm annually (p = 0.040) was found to be significant.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

The aims of the present study were: (a) to describe the prevalence of helminth infections from pooled faecal samples from sheep flocks across Greece and (b) to evaluate flock-related factors potentially associated with the presence of these infections in the flocks. An extensive countrywide study was performed on 325 sheep farms throughout Greece; faecal samples were collected from ewes and processed for the identification of helminth parasites. Helminths were detected in samples from 92.9% of flocks; these included Dicrocoelium dendriticum (16.7% of flocks), Fasciola hepatica (0.6%), Paramphistomum cervi (2.2%), Moniezia spp. (18.8%), Trichostrongylidae (85.5%), Nematodirus spp. (18.8%), Strongyloides papillosus (7.1%), Trichuris spp. (20.0%) and lungworms (17.8%). Mean Trichostrongylidae counts across all flocks in the study were 215 epg. Specifically, for Trichostrongylidae, there were differences between flocks that had or had not received anthelmintics during the two months prior to sampling, as well as between flocks located in various areas of the country. In multivariable analyses, for the outcome \'high (>300) epg counts in faecal samples\', the month into the lactation period at sampling and the application of reproductive control practices on the farm emerged as significant factors; for the outcome \'high proportion (>63%) of Teladorsagia spp. in faecal samples\', the availability of straw bedding emerged as a significant factor; and for the outcome \'high proportion (>63%) of Haemonchus contortus in faecal samples\', the age of the farmer emerged as a significant factor. For the outcome \'presence of Trichuris spp. in faecal samples\', the provision of finished feed (concentrate) to animals emerged as a significant factor, whilst, finally, for the outcomes \'presence of D. dendriticum in faecal samples\' and \'presence of lungworms in faecal samples,\' no significant factors emerged.

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites.

Global warming and displacement of vectors and wild and domestic hosts by humans greatly change host-parasite interactions and parasite transmission rates. Thelazia callipaeda Railliet and Henry, 1910 (Spirurida: Thelaziidae) is a zoonotic parasite rapidly colonizing Europe from its Asian native range. This nematode is vectored by Phortica flies and may cause ocular disorders, such as keratitis and corneal ulcers, in wild mammals, dogs, cats and humans. With the aim of detecting possible threats for the European wildcat (Felis silvestris) in one of its last Mediterranean strongholds, we initiated in 2017 a long-term monitoring program in Sierra Arana (southeastern Spain), which includes exploring its epidemiological and spatial relationships with domestic cats (Felis catus). During routine medical check-ups, we detected ocular nematodes in 3 of 17 (17.6%) wildcats and 1 of 23 (4.3%) domestic cats tested, confirmed to be T. callipaeda by microscopic and molecular analyses. This nematode species was first detected in the study area in 2021. To our knowledge, this is the first case of infection in wildcats in Spain. Through telemetry, we detected little spatial overlap between the home ranges of wildcats and domestic cats, which seems to be sufficient to facilitate the inter-specific transmission of T. callipaeda. Our findings suggest that this pathogen could be a new threat to the wildcat, a locally endangered wild felid. Further eco-epidemiological monitoring and sanitary control of domestic cats will be strongly needed, especially given the ongoing global warming scenario.

Clostridioide difficile is the leading cause of diarrhea disease worldwide and is a CDC-designated urgent threat level pathogen. Mammalian models are commonly utilized as gold standard to study the pathogenesis of C. difficile infection (CDI); however, alternatives are needed due to cost, higher throughput ability, and mammalian animal ethics. Nonmammalian models such as great wax worm, nematode, fruit fly, and zebrafish have been used as CDI models. This review provides a comprehensive summary of nonmammalian models used to study CDI. Multiple studies were identified using these models to study C. difficile infection, pathogenicity, colonization, host immunity, and therapy. Translational outcomes and strength and weakness of each nonmammalian model are discussed.

Unconventional myosins are a superfamily of actin-based motor proteins that perform a number of roles in fundamental cellular processes, including (but not limited to) intracellular trafficking, cell motility, endocytosis, exocytosis and cytokinesis. 40 myosins genes have been identified in humans, which belong to different 12 classes based on their domain structure and organisation. These genes are widely expressed in different tissues, and mutations leading to loss of function are associated with a wide variety of pathologies while over-expression often results in cancer. Caenorhabditis elegans (C. elegans) is a small, free-living, non-parasitic nematode. ~38% of the genome of C. elegans has predicted orthologues in the human genome, making it a valuable tool to study the function of human counterparts and human diseases. To date, 8 unconventional myosin genes have been identified in the nematode, from 6 different classes with high homology to human paralogues. The hum-1 and hum-5 (heavy chain of an unconventional myosin) genes encode myosin of class I, hum-2 of class V, hum-3 and hum-8 of class VI, hum-6 of class VII and hum-7 of class IX. The hum-4 gene encodes a high molecular mass myosin (307 kDa) that is one of the most highly divergent myosins and is a member of class XII. Mutations in many of the human orthologues are lethal, indicating their essential properties. However, a functional characterisation for many of these genes in C. elegans has not yet been performed. This article reviews the current knowledge of unconventional myosin genes in C. elegans and explores the potential use of the nematode to study the function and regulation of myosin motors to provide valuable insights into their role in diseases.

The southern root-knot nematode (SRKN; Meloidogyne incognita) is a typical parasitic nematode that affects sweetpotato [Ipomoea batatas (L.) Lam.], causing a significant decrease in crop yield and commercial value. In Japan, the SRKN is classified into 10 races: SP1-SP5, SP6-1, SP6-2, and SP7-SP9, with the dominant race differing according to the cultivation area. Soil insecticides have previously been used to reduce the soil density of SRKNs; however, this practice is both costly and labor intensive. Therefore, the development of SRKN-resistant sweetpotato lines and cultivars is necessary. However, due to the complexity of polyploid inheritance and the highly heterogeneous genomic composition of sweetpotato, genetic information and research for this species are significantly lacking compared to those for other major diploid crop species. In this study, we utilized the recently developed genome-wide association approach, which uses multiple-dose markers to assess autopolyploid species. We performed an association analysis to investigate resistance toward SRKN-SP2, which is the major race in areas with high sweetpotato production in Japan. The segregation ratio of resistant and susceptible lines in the F1 mapping population derived from the resistant \"J-Red\" and susceptible \"Choshu\" cultivars was fitted to 1: 3, suggesting that resistance to SP2 may be regulated by two loci present in the simplex. By aligning the double digest restriction-site associated DNA sequencing reads to the published Ipomoea trifida reference sequence, 46,982 single nucleotide polymorphisms (SNPs) were identified (sequencing depth > 200). The association study yielded its highest peak on chromosome 7 (Chr07) and second highest peak on chromosome 3 (Chr03), presenting as a single-dose in both loci. Selective DNA markers were developed to screen for resistant plants using the SNPs identified on Chr03 and Chr07. Our results showed that SRKN-SP2-resistant plants were selected with a probability of approximately 70% when combining the two selective DNA markers. This study serves as a model for the identification of genomic regions that control agricultural traits and the elucidation of their effects, and is expected to greatly advance marker-assisted breeding and association studies in polyploid crop species.

BACKGROUND: The peach fruit fly, Bactrocera zonata, and the Oriental fruit fly, B. dorsalis (Diptera: Tephritidae), are economically important fruit fly species in various regions of the world. We evaluated the effects of separate and combined applications of the entomopathogenic fungi (EPF) Beauveria bassiana (WG-18) and Metarhizium anisopliae (WG-02), and the entomopathogenic nematodes (EPNs) Heterorhabditis bacteriophora (VS strain) and Steinernema carpocapsae (ALL strain) against larvae, pupae and pharate adults, of B. zonata and B. dorsalis under laboratory, glasshouse and field cage conditions.
RESULTS: Combined applications of EPF and EPNs produced greater mortality than individual treatments under all conditions. Against both species, the combination of B. bassiana and H. bacteriophora consistently exerted strong effects that were similar to the combined application of B. bassiana and S. carpocapsae whereas M. anisopliae applied with S. carpocapsae was least effective in all combinations. In a laboratory bioassay, synergistic interactions were detected between B. bassiana and H. bacteriophora applied against larvae and pharate adults of both fly species, between B. bassiana and S. carpocapsae against larvae of both species and pharate adults of B. zonata, and between M. anisopliae and H. bacteriophora against B. zonata larvae. Other combined treatments resulted in additive effects, especially against fly pupae. In a potted soil bioassay, there were only additive interactions in all combinations against different stages of both flies. The 3rd instar of both flies was more susceptible than pharate adult and pupal stages. Additive interactions between EPNs and EPF were detected in the glasshouse against 3rd instars and pupae, and under field conditions against 3rd instars of both fly species.
CONCLUSIONS: These results indicate how particular combinations of entomopathogenic fungi and nematodes could be deployed in integrated pest management of tephritid fruit flies in orchard agro-ecosystems. © 2022 Society of Chemical Industry.