Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona.

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes. IMPORTANCE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

A diverse microbial community and common core microbiota associated with the gonad of female Parascaris spp.

The microbiome plays an important role in health, where changes in microbiota composition can have significant downstream effects within the host, and host-microbiota relationships can be exploited to affect health outcomes. Parasitic helminths affect animals globally, but an exploration of their microbiota has been limited, despite the development of anti-Wolbachia drugs to help control infections with some filarial nematodes. The equine ascarids, Parascaris spp., are considered the most pathogenic nematodes affecting juvenile horses and are also the only ascarid parasite to have developed widespread anthelmintic resistance. The aim of this study was to characterize the microbiota of this helminth, focusing on the female gonad, determine a core microbiota for this organ, identify bacterial species, and show bacterial localization to the female gonad via in situ hybridization (ISH). A total of 22 gonads were isolated from female Parascaris spp. collected from three foals, and 9 female parasites were formalin-fixed and paraffin-embedded for ISH. Next-generation sequencing was performed using V3-V4 primers as well as the Swift Amplicon™ 16S+ ITS Panel. Overall, ten genera were identified as members of the Parascaris spp. female gonad and twelve bacterial species were identified. The most prevalent genus was Mycoplasma, followed by Reyranella, and there were no differences in alpha diversity between parasites from different horses. Specific eubacteria staining was identified in both the intestine and within the gonad using ISH. Overall, this study provided in-depth information regarding the female Parascaris spp. microbiota and was the first to identify the core microbiota within a specific parasite organ.

Abnormal mare behaviour is rarely associated with changes in hormonal markers of granulosa cell tumours: A retrospective study.

BACKGROUND: Abnormal or undesired mare behaviours are often assumed to be associated with ovarian abnormalities. OBJECTIVES: We aimed to determine the incidence of abnormal behaviours and their association with concentrations of one or more ovarian hormones associated with a granulosa cell tumour (GCT). STUDY DESIGN: Retrospective descriptive. METHODS: A total of 2914 hormonal profile samples submitted with the words behave, behaviour, or behaving in the submission history were analysed. The association between reported abnormal behaviours and concentrations of testosterone, anti-Müllerian hormone (AMH), inhibins and inhibin-B were assessed. Statistical analysis was performed using a Chi-squared test of association. RESULTS: Of the 2914 cases that were submitted due to behaviour issues, 2506 (86%) did not have any of the measured hormones reach GCT-like concentrations. The remaining 408 cases had either one (63%), two (25.5%), or three (11.5%) hormones with concentrations consistent with those from confirmed GCT cases. Testosterone had the lowest percent of GCT-like values among the cases (7.7%), compared with AMH (9.4%), inhibins (9.6%) and inhibin B (8.7%). Stallion-like behaviour was significantly associated with increased concentrations of all four hormones. In contrast, aggression, oestrous and other abnormal behaviours were significantly less likely to be associated with increased concentrations of the hormones. MAIN LIMITATIONS: Retrospective study, using sample submission history. CONCLUSION: Overall, the abnormal behaviours among mares, except the stallion-like behaviour, were not associated with increased ovarian hormones. These results highlight the common misassumption about the involvement of the ovaries in 'abnormal behaviours' or 'undesirable behaviours' of mares.

The microbial community associated with Parascaris spp. infecting juvenile horses.

BACKGROUND: Parasitic nematodes, including large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarids, Parascaris spp., are important parasites of juvenile horses and the first ascarids to develop widespread anthelmintic resistance. The microbiota has been shown to be an important factor in the fitness of many organisms, including parasitic nematodes, where endosymbiotic Wolbachia have been exploited for treatment of filariasis in humans. METHODS: This study used short-read 16S rRNA sequences and Illumina sequencing to characterize and compare microbiota of whole worm small intestinal stages and microbiota of male and female intestines and gonads. Diversity metrics including alpha and beta diversity, and the differential abundance analyses DESeq2, ANCOM-BC, corncob, and metagenomeSeq were used for comparisons. RESULTS: Alpha and beta diversity of whole worm microbiota did not differ significantly between groups, but Simpson alpha diversity was significantly different between female intestine (FI) and male gonad (MG) (P= 0.0018), and Shannon alpha diversity was significantly different between female and male gonads (P = 0.0130), FI and horse jejunum (HJ) (P = 0.0383), and FI and MG (P= 0.0001). Beta diversity (Fig. 2B) was significantly different between female and male gonads (P = 0.0006), male intestine (MI) and FG (P = 0.0093), and MG and FI (P = 0.0041). When comparing organs, Veillonella was differentially abundant for DESeq2 and ANCOM-BC (p < 0.0001), corncob (P = 0.0008), and metagenomeSeq (P = 0.0118), and Sarcina was differentially abundant across four methods (P < 0.0001). Finally, the microbiota of all individual Parascaris spp. specimens were compared to establish shared microbiota between groups. CONCLUSIONS: Overall, this study provided important information regarding the Parascaris spp. microbiota and provides a first step towards determining whether the microbiota may be a viable target for future parasite control options.

Transcriptomic and histochemical analysis reveal the complex regulatory networks in equine chorioallantois during spontaneous term labor†.

The equine chorioallantois (CA) undergoes complex physical and biochemical changes during labor. However, the molecular mechanisms controlling these changes are still unclear. Therefore, the current study aimed to characterize the transcriptome of equine CA during spontaneous labor and compare it with that of normal preterm CA. Placental samples were collected postpartum from mares with normal term labor (TL group, n = 4) and from preterm not in labor mares (330 days GA; PTNL group, n = 4). Our study identified 4137 differentially expressed genes (1820 upregulated and 2317 downregulated) in CA during TL as compared with PTNL. TL was associated with the upregulation of several proinflammatory mediators (MHC-I, MHC-II, NLRP3, CXCL8, and MIF). Also, TL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP2, MMP3, and MMP9) with subsequent extracellular matrix degradation and apoptosis, as reflected by upregulation of several apoptosis-related genes (ATF3, ATF4, FAS, FOS, and BIRC3). In addition, TL was associated with downregulation of 21 transcripts coding for collagens. The upregulation of proteases, along with the downregulation of collagens, is believed to be implicated in separation and rupture of the CA during TL. Additionally, TL was associated with downregulation of transcripts coding for proteins essential for progestin synthesis (SRD5A1 and AKR1C1) and angiogenesis (VEGFA and RTL1), as well as upregulation of prostaglandin synthesis-related genes (PTGS2 and PTGES), which could reflect the physiological switch in placental endocrinology and function during TL. In conclusion, our findings revealed the equine CA gene expression signature in spontaneous labor at term, which improves our understanding of the molecular mechanisms triggering labor.

Parental bias in expression and interaction of genes in the equine placenta.

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Transcriptomic analysis of equine placenta reveals key regulators and pathways involved in ascending placentitis†.

Improved understanding of the molecular mechanisms underlying ascending equine placentitis holds the potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor. The current study characterized the equine placental transcriptome (chorioallantois [CA] and endometrium [EN]) during placentitis (placentitis group, n = 6) in comparison to gestationally-matched controls (control group, n = 6). Transcriptome analysis identified 2953 and 805 differentially expressed genes in CA and EN during placentitis, respectively. Upstream regulator analysis revealed the central role of toll-like receptors (TLRs) in triggering the inflammatory signaling, and consequent immune-cell chemotaxis. Placentitis was associated with the upregulation of matrix metalloproteinase (MMP1, MMP2, and MMP9) and apoptosis-related genes such as caspases (CASP3, CASP4, and CASP7) in CA. Also, placentitis was associated with downregulation of transcripts coding for proteins essential for placental steroidogenesis (SRD5A1 and AKR1C1), progestin signaling (PGRMC1 and PXR) angiogenesis (VEGFA, VEGFR2, and VEGFR3), and nutrient transport (GLUT12 and SLC1A4), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could explain placental insufficiency during placentitis. Placentitis was also associated with aberrant expression of several placenta-regulatory genes, such as PLAC8, PAPPA, LGALS1, ABCG2, GCM1, and TEPP, which could negatively affect placental functions. In conclusion, our findings revealed for the first time the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during equine placentitis, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Pixel by pixel: real-time observation and quantification of passive flotation speeds of three common equine endoparasite egg types.

The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotation protocols exist, little is known about the characteristics of these eggs with respect to their movement through a flotation solution. In this study, we have demonstrated a novel method for the observation and quantification of microscopic (65-100 µm) objects as they experience unassisted flotation. This also represents, to our knowledge for the first time, that the flotation of parasite eggs has been observed and their movement characteristics quantified as they float through solution. Particle tracking and video analysis software were utilised to automatically detect and track the movement of individual eggs as they floated. Three 30 s videos and one 2 min video of each egg type were analysed. If the first 30 s of video were discounted, the differences in mean flotation speed among all videos was statistically significant between egg types (P = 0.0004). Strongyle type eggs (n = 201) moved the fastest with a mean 51.08 µm/s (95% confidence interval: 47.54-54.62). This was followed by Parascaris spp. (n = 131) and Anoplocephala perfoliata eggs (n = 322), with mean speeds of 44.43 µm/s (95% confidence interval: 39.47-49.4) and 31.11 µm/s (95% confidence interval: 29.6-32.61), respectively. This method for evaluating the mean speed of passive flotation may represent a first step towards further optimizing fecal egg flotation and be of interest to parasitologists and veterinary practitioners.

Landscape of Overlapping Gene Expression in the Equine Placenta.

Increasing evidence suggests that overlapping genes are much more common in eukaryotic genomes than previously thought. These different-strand overlapping genes are potential sense-antisense (SAS) pairs, which might have regulatory effects on each other. In the present study, we identified the SAS loci in the equine genome using previously generated stranded, paired-end RNA sequencing data from the equine chorioallantois. We identified a total of 1261 overlapping loci. The ratio of the number of overlapping regions to chromosomal length was numerically higher on chromosome 11 followed by chromosomes 13 and 12. These results show that overlapping transcription is distributed throughout the equine genome, but that distributions differ for each chromosome. Next, we evaluated the expression patterns of SAS pairs during the course of gestation. The sense and antisense genes showed an overall positive correlation between the sense and antisense pairs. We further provide a list of SAS pairs with both positive and negative correlation in their expression patterns throughout gestation. This study characterizes the landscape of sense and antisense gene expression in the placenta for the first time and provides a resource that will enable researchers to elucidate the mechanisms of sense/antisense regulation during pregnancy.

Histologically, immunohistochemically, ultrastructurally, and molecularly confirmed neosporosis abortion in an aborted equine fetus.

Neosporosis is a common cause of abortion in cattle worldwide but is rare in horses. Here, the first case of histologically, ultrastructurally, immunohistochemically, and molecularly confirmed equine abortion caused by neosporosis is reported. Samples of lung, heart, liver, skeletal muscle, tongue, brain, and the placenta from a female fetus aborted at 280 days of gestation were fixed in formalin and submitted for diagnosis. Histologically, there was disseminated neosporosis with severe lesions in lungs, liver and the heart. Protozoal tachyzoites in all tissues reacted with polyclonal anti-Neospora caninum rabbit antibodies. Transmission electron microscopic observation on lung tissue revealed tachyzoites consistent with Neospora, including many rhoptries. Polymerase-chain reaction (PCR) using primers designed to amplify the rRNA gene internal transcribed spacer 1 (ITS1) of the Sarcocystidae was performed on DNA extracted from fetal tissues. Comparison of the ITS1 amplified from the foal tissue to sequences available in GenBank revealed 100% sequence identity to the ITS1 from three isolates of Neospora hughesi.

Determination of the specific gravity of eggs of equine strongylids, Parascaris spp., and Anoplocephala perfoliata.

Given the ever-increasing levels of anthelmintic resistance in livestock parasites globally, it is recommended to use parasite fecal egg counts to make treatment decisions and to evaluate treatment efficacy. The consensus in equine parasitology is to use a flotation medium with a specific gravity (SG) of ≥ 1.20 to float the main parasite egg types of interest in egg counting techniques. However, the density of common equine endoparasite eggs has been sparsely investigated. Equine tapeworm eggs are known to be particularly difficult to determine and count in fecal samples. It is unknown whether this could be because of differences in egg density. The aim of this study was to provide estimates of relative densities for equine ascarid, strongyle, and tapeworm eggs. Six aqueous glucose-salt solutions with specific gravities ranging from 1.06 to 1.16 were made and placed from most to least dense into thirteen 15 mL centrifuge tubes. Concentrated aqueous suspensions of the three types of endoparasite eggs were placed on top of each tube. These tubes were then centrifuged at 800 g for 20 min and each layer of flotation solution was carefully pipetted and transferred to a McMaster egg counting slide. Egg type and count were recorded for each specific gravity layer. Each egg was assigned a specific gravity based on the specific gravity layer it was observed in. In a second trial of this study, five similar flotation media were made ranging from 1.02 to 1.10 and were used in four subsequent replicates. In total between the two trials, the mean egg SGs of Anoplocephala perfoliata (n = 3811), Parascaris spp. (n = 3478), and strongylid type eggs (n = 9291) were 1.0636 (95% confidence interval (CI): 1.0629-1.0642), 1.0903 (95% CI: 1.0897-1.0909), and 1.0453 (95% CI: 1.0448-1.0458), respectively. The three egg types were statistically different from each other (p < 0.0001). This is the first time that the specific gravity of equine strongylid and Anoplocephala perfoliata eggs has been determined. With a tapeworm egg density demonstrated to be between that of strongylids and Parascaris spp., the poor recovery of tapeworm eggs in equine fecal samples must have other explanations.

Anthelmintic therapy of equine cyathostomin nematodes - larvicidal efficacy, egg reappearance period, and drug resistance.

Cyathostomins are ubiquitous in grazing horses across the world, and anthelmintic resistance has been reported with increasing levels over past decades. The aims of the present study were (i) to investigate the efficacy against encysted larval stages of moxidectin (0.4 mg/kg) and fenbendazole (10 mg/kg daily for five consecutive days) and compare these regimens at 2 and 5 weeks post-treatment, (ii) to investigate individual cyathostomin species associated with shortened egg reappearance periods, and (iii) to document species exhibiting decreased susceptibility to the evaluated compounds. Thirty-six ponies were allocated to treatment groups with half euthanatized 2 weeks post-treatment, and the remainder necropsied after 5 weeks. Luminal and mucosal worm counts were conducted and strongyle egg counts were determined at weekly intervals. At 2 weeks, mean reductions of early L3s were 50.4% and 73.8% for fenbendazole and moxidectin, respectively. At 5 weeks, the respective efficacies were 51.3% and 71.8%. Two week efficacies against late L3s and L4s (LL3s/L4s) were 70.8% and 74.6% for fenbendazole and moxidectin, respectively, whereas very low numbers were found in all three groups at 5 weeks. None of the mucosal counts were significantly different between treatment groups. Fenbendazole and moxidectin reduced luminal worm counts by 93.2% and 98.3% at 2 weeks following administration, with moxidectin group adult counts being significantly lower than the other two groups (P < 0.0001). Both treatment groups had increased counts 3 weeks later (P = 0.0415). A moxidectin ERP of 4 weeks was associated with surviving luminal L4s, and adult species contributing to this were Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus ashworthi and Cylicocyclus nassatus. This study documented (i) larvicidal efficacy of fenbendazole much lower than historical standards, (ii) survival of luminal immatures (L4) following moxidectin administration, and (iii) new information about cyathostomin species associated with these phenomena.