Dentition patterns and molecular diversity of Mastophorus muris (Gmelin, 1790) (Nematoda: Spiruroidea) support a host-associated subdivision.

Mastophorus muris (Gmelin, 1790) is a globally distributed parasitic nematode of broad range mammals. The taxonomy within the genus Mastophorus and the cryptic diversity among the genus are controversial among taxonomists. This study provides a detailed morphological description of M. muris from Mus musculus combined with a molecular phylogenetic approach. Moreover, descriptions and molecular data of M. muris from non-Mus rodents and wildcats complement our findings and together provide new insights into their taxonomy. The analysis of M. muris was based on light microscopy and scanning electron microscopy. The morphological description focused on the dentition pattern of the two trilobed pseudolabia. Additionally, we described the position of the vulva, arrangement of caudal pairs of papillae, spicules and measured specimens from both sexes and the eggs. For the molecular phylogenetic approach, we amplified the small subunit ribosomal RNA gene and the internal transcribed spacer, and the cytochrome c oxidase subunit 1. Mastophorus morphotypes based on dentition patterns and phylogenetic clustering indicate a subdivision of the genus in agreement with their host. We recognize two groups without a change to formal taxonomy: One group including those specimens infecting Mus musculus, and the second group including organisms infecting non-Mus rodents. Our genetic and morphological data shed light into the cryptic diversity within the genus Mastopohorus. We identified two host-associated groups of M. muris. The described morphotypes and genotypes of M. muris allow a consistent distinction between host-associated parasites.

Aberrant microbiomes are associated with increased antibiotic resistance gene load in hybrid mice.

Antibiotic resistance is a priority public health problem resulting from eco-evolutionary dynamics within microbial communities and their interaction at a mammalian host interface or geographical scale. The links between mammalian host genetics, bacterial gut community, and antimicrobial resistance gene (ARG) content must be better understood in natural populations inhabiting heterogeneous environments. Hybridization, the interbreeding of genetically divergent populations, influences different components of the gut microbial communities. However, its impact on bacterial traits such as antibiotic resistance is unknown. Here, we present that hybridization might shape bacterial communities and ARG occurrence. We used amplicon sequencing to study the gut microbiome and to predict ARG composition in natural populations of house mice (Mus musculus). We compared gastrointestinal bacterial and ARG diversity, composition, and abundance across a gradient of pure and hybrid genotypes in the European House Mouse Hybrid Zone. We observed an increased overall predicted richness of ARG in hybrid mice. We found bacteria-ARG interactions by their co-abundance and detected phenotypes of extreme abundances in hybrid mice at the level of specific bacterial taxa and ARGs, mainly multidrug resistance genes. Our work suggests that mammalian host genetic variation impacts the gut microbiome and chromosomal ARGs. However, it raises further questions on how the mammalian host genetics impact ARGs via microbiome dynamics or environmental covariates.

Host weight, seasonality and anthropogenic factors contribute to parasite community differences between urban and rural foxes.

Pathogens often occur at different prevalence along environmental gradients. This is of particular importance for gradients of anthropogenic impact such as rural-urban transitions presenting a changing interface between humans and wildlife. The assembly of parasite communities is affected by both the external environmental conditions and individual host characteristics. Hosts with low body weight (smaller individuals or animals with poor body condition) might be more susceptible to infection. Furthermore, parasites' mode of transmission might affect their occurrence: rural environments with better availability of intermediate hosts might favour trophic transmission, while urban environments, typically with dense definitive host populations, might favour direct transmission. We here study helminth communities (141 intestinal samples) within the red fox (Vulpes vulpes), a synanthropic host, using DNA metabarcoding of multiple marker genes. We analysed the effect of urbanisation, seasonality and host-intrinsic (weight, sex) variables on helminth communities. Helminth species richness increased in foxes with lower body weight and in winter and spring. Season and urbanisation, however, had strong effects on the community composition, i.e., on the identity of the detected species. Surprisingly, transmission in two-host life cycles (trophic transmission) was more pronounced in urban Berlin than in rural Brandenburg. This disagrees with the prevailing hypothesis that trophically transmitted helminths are less prevalent in urban areas than in rural areas. Generally, co-infestations with multiple helminths and high infection intensity are associated with lighter (younger, smaller or low body condition) animals. Both host-intrinsic traits and environmental drivers together shape parasite community composition and turnover along urban-rural gradients.

Towards monitoring of antimicrobial resistance in the environment: For what reasons, how to implement it, and what are the data needs?

Antimicrobial resistance (AMR) is a global threat to human and animal health and well-being. To understand AMR dynamics, it is important to monitor resistant bacteria and resistance genes in all relevant settings. However, while monitoring of AMR has been implemented in clinical and veterinary settings, comprehensive monitoring of AMR in the environment is almost completely lacking. Yet, the environmental dimension of AMR is critical for understanding the dissemination routes and selection of resistant microorganisms, as well as the human health risks related to environmental AMR. Here, we outline important knowledge gaps that impede implementation of environmental AMR monitoring. These include lack of knowledge of the 'normal' background levels of environmental AMR, definition of high-risk environments for transmission, and a poor understanding of the concentrations of antibiotics and other chemical agents that promote resistance selection. Furthermore, there is a lack of methods to detect resistance genes that are not already circulating among pathogens. We conclude that these knowledge gaps need to be addressed before routine monitoring for AMR in the environment can be implemented on a large scale. Yet, AMR monitoring data bridging different sectors is needed in order to fill these knowledge gaps, which means that some level of national, regional and global AMR surveillance in the environment must happen even without all scientific questions answered. With the possibilities opened up by rapidly advancing technologies, it is time to fill these knowledge gaps. Doing so will allow for specific actions against environmental AMR development and spread to pathogens and thereby safeguard the health and wellbeing of humans and animals.

Amplicon sequencing allows differential quantification of closely related parasite species: an example from rodent Coccidia (Eimeria).

BACKGROUND: Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended. METHODS: We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population. RESULTS: We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance. CONCLUSIONS: We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.

Resilience and stability of the CF- intestinal and respiratory microbiome during nutritional and exercise intervention.

BACKGROUND: Impaired respiratory and intestinal microbiome composition is linked to cystic fibrosis lung disease severity. In people with cystic fibrosis (pwCF), regular exercise is recommended to delay disease progression and preserve a stable lung function. An optimal nutritional status is vital for best clinical outcomes. Our study investigated whether regular and monitored exercise and nutritional support promotes CF microbiome health. METHODS: A personalized nutrition and exercise program promoted nutritional intake and physical fitness in 18 pwCF for 12 months. Throughout the study, patients performed strength and endurance training monitored by a sports scientist via an internet platform. After three months, food supplementation with Lactobacillus rhamnosus LGG was introduced. Nutritional status and physical fitness were assessed before the study started, after three and nine months. Sputum and stool were collected, and microbial composition was analyzed by 16S rRNA gene sequencing. RESULTS: Sputum and stool microbiome composition remained stable and highly specific to each patient during the study period. Disease-associated pathogens dominated sputum composition. Lung disease severity and recent antibiotic treatment had the highest impact on taxonomic composition in stool and sputum microbiome. Strikingly, the long-term antibiotic treatment burden had only a minor influence. CONCLUSION: Despite the exercise and nutritional intervention, respiratory and intestinal microbiomes proved to be resilient. Dominant pathogens drove the composition and functionality of the microbiome. Further studies are required to understand which therapy could destabilize the dominant disease-associated microbial composition of pwCF.

Guts within guts: the microbiome of the intestinal helminth parasite Ascaris suum is derived but distinct from its host.

BACKGROUND: Intestinal helminths are extremely prevalent among humans and animals. In particular, intestinal roundworms affect more than 1 billion people around the globe and are a major issue in animal husbandry. These pathogens live in intimate contact with the host gut microbiota and harbor bacteria within their own intestines. Knowledge of the bacterial host microbiome at the site of infection is limited, and data on the parasite microbiome is, to the best of our knowledge, non-existent. RESULTS: The intestinal microbiome of the natural parasite and zoonotic macropathogen, Ascaris suum was analyzed in contrast to the diversity and composition of the infected host gut. 16S sequencing of the parasite intestine and host intestinal compartments showed that the parasite gut has a significantly less diverse microbiome than its host, and the host gut exhibits a reduced microbiome diversity at the site of parasite infection in the jejunum. While the host's microbiome composition at the site of infection significantly determines the microbiome composition of its parasite, microbial signatures differentiate the nematodes from their hosts as the Ascaris intestine supports the growth of microbes that are otherwise under-represented in the host gut. CONCLUSION: Our data clearly indicate that a nematode infection reduces the microbiome diversity of the host gut, and that the nematode gut represents a selective bacterial niche harboring bacteria that are derived but distinct from the host gut. Video Abstract.

DNA-based quantification and counting of transmission stages provides different but complementary parasite load estimates: an example from rodent coccidia (Eimeria).

BACKGROUND: Counting parasite transmission stages in faeces is the classical measurement to quantify "parasite load". DNA-based quantifications of parasite intensities from faecal samples are relatively novel and often validated against such counts. When microscopic and molecular quantifications do not correlate, it is unclear whether oocyst counts or DNA-based intensity better reflects biologically meaningful concepts. Here, we investigate this issue using the example of Eimeria ferrisi (Coccidia), an intracellular parasite of house mice (Mus musculus). METHODS: We performed an infection experiment of house mice with E. ferrisi, in which the intensity of infection correlates with increased health impact on the host, measured as temporary weight loss during infection. We recorded the number of parasite transmissive stages (oocysts) per gram of faeces (OPG) and, as a DNA-based measurement, the number of Eimeria genome copies per gram of faeces for 10 days post-infection (dpi). We assessed weight loss relative to the day of experimental infection as a proxy of host health and evaluated whether DNA or oocyst counts are better predictors of host health. RESULTS: Absolute quantification of Eimeria DNA and oocyst counts showed similar but slightly diverging temporal patterns during 10 dpi. We detected Eimeria DNA earlier than the first appearance of oocysts in faeces. Additionally, Eimeria OPGs within each dpi did not explain parasite DNA intensity. Early dpi were characterized by high DNA intensity with low oocyst counts, while late infections showed the opposite pattern. The intensity of Eimeria DNA was consistently a stronger predictor of either maximal weight loss (1 value per animal during the infection course) or weight loss on each day during the experiment when controlling for between-dpi and between-individual variance. CONCLUSIONS: Eimeria ferrisi oocyst counts correlate weakly with parasite intensity assessed through DNA quantification. DNA is likely partially derived from life-cycle stages other than transmissive oocysts. DNA-based intensities predict health outcomes of infection for the host more robustly than counts of transmissive stages. We conclude that DNA-based quantifications should not necessarily require validation against counts of transmissive stages. Instead, DNA-based load estimates should be evaluated as complementary sources of information with potential specific biological relevance for each host-parasite system.

Phylogeography and population differentiation in Hepatozoon canis (Apicomplexa: Hepatozoidae) reveal expansion and gene flow in world populations.

BACKGROUND: Hepatozoon canis is a protozoan transmitted to dogs and other wild carnivores by the ingestion of ticks containing mature oocysts and is considered the principal cause of canine hepatozoonosis in the world. Here, we examined ribosomal RNA 18S gene sequence variation to determine the genetic differences and phylogeographic diversity of H. canis from various geographical areas around the world. METHODS: We used 550 publicly available sequences of H. canis from 46 countries to assess haplotype relationships, geographical structure, genetic diversity indices, and relationships among populations. We performed neutrality tests and pairwise comparisons of fixation index (FST) values between groups and pairwise comparisons of FST values between populations. To determine whether populations are structured, analyses of molecular variance (AMOVAs) and spatial analysis of molecular variance (SAMOVA) were performed. RESULTS: The dataset of H. canis yielded 76 haplotypes. Differentiation among populations indicated that there is no phylogeographical structure (GST = 0.302 ± 0.0475). Moreover, when samples were grouped by continents a significant FST was obtained, meaning that populations were genetically differentiated. The AMOVA showed that 57.4% of the genetic variation was explained by differences within populations when all locations were treated as a single group and revealed that there is no population structure when populations are grouped into two, three, and four groups (FCT, p > 0.05), suggesting that dispersal between populations is high. SAMOVA revealed significant FCT values for groups K = 5. The Tajima's D and Fu's Fs show that populations have undergone recent expansion, and the mismatch distribution analysis showed population expansion (multimodal distribution). CONCLUSIONS: The current molecular data confirmed that H. canis does not show phylogeographic or population structure. The haplotypes exhibit low genetic differentiation, suggesting a recent expansion due to gene flow among populations. These results provide pivotal information required for future detailed population genetic analysis or to establish control strategies of this parasite.

Generalist Eimeria species in rodents: Multilocus analyses indicate inadequate resolution of established markers.

Intracellular parasites of the genus Eimeria are described as tissue/host-specific. Phylogenetic classification of rodent Eimeria suggested that some species have a broader host range than previously assumed. We explore whether Eimeria spp. infecting house mice are misclassified by the most widely used molecular markers due to a lack of resolution, or whether, instead, these parasite species are indeed infecting multiple host species.With the commonly used markers (18S/COI), we recovered monophyletic clades of E. falciformis and E. vermiformis from Mus that included E. apionodes identified in other rodent host species (Apodemus spp., Myodes glareolus, and Microtus arvalis). A lack of internal resolution in these clades could suggest the existence of a species complex with a wide host range infecting murid and cricetid rodents. We question, however, the power of COI and 18S markers to provide adequate resolution for assessing host specificity. In addition to the rarely used marker ORF470 from the apicoplast genome, we present multilocus genotyping as an alternative approach. Phylogenetic analysis of 35 nuclear markers differentiated E. falciformis from house mice from isolates from Apodemus hosts. Isolates of E. vermiformis from Mus are still found in clusters interspersed with non-Mus isolates, even with this high-resolution data.In conclusion, we show that species-level resolution should not be assumed for COI and 18S markers in coccidia. Host-parasite cospeciation at shallow phylogenetic nodes, as well as contemporary coccidian host ranges more generally, is still open questions that need to be addressed using novel genetic markers with higher resolution.

Coupling between tolerance and resistance for two related Eimeria parasite species.

Resistance (host capacity to reduce parasite burden) and tolerance (host capacity to reduce impact on its health for a given parasite burden) manifest two different lines of defense. Tolerance can be independent from resistance, traded off against it, or the two can be positively correlated because of redundancy in underlying (immune) processes. We here tested whether this coupling between tolerance and resistance could differ upon infection with closely related parasite species. We tested this in experimental infections with two parasite species of the genus Eimeria. We measured proxies for resistance (the (inverse of) number of parasite transmission stages (oocysts) per gram of feces at the day of maximal shedding) and tolerance (the slope of maximum relative weight loss compared to day of infection on number of oocysts per gram of feces at the day of maximal shedding for each host strain) in four inbred mouse strains and four groups of F1 hybrids belonging to two mouse subspecies, Mus musculus domesticus and Mus musculus musculus. We found a negative correlation between resistance and tolerance against Eimeria falciformis, while the two are uncoupled against Eimeria ferrisi. We conclude that resistance and tolerance against the first parasite species might be traded off, but evolve more independently in different mouse genotypes against the latter. We argue that evolution of the host immune defenses can be studied largely irrespective of parasite isolates if resistance-tolerance coupling is absent or weak (E. ferrisi) but host-parasite coevolution is more likely observable and best studied in a system with negatively correlated tolerance and resistance (E. falciformis).

Intensity of infection with intracellular Eimeria spp. and pinworms is reduced in hybrid mice compared to parental subspecies.

Genetic diversity in animal immune systems is usually beneficial. In hybrid recombinants, this is less clear, as the immune system could also be impacted by genetic conflicts. In the European house mouse hybrid zone, the long-standing impression that hybrid mice are more highly parasitized and less fit than parentals persists despite the findings of recent studies. Working across a novel transect, we assessed infections by intracellular protozoans, Eimeria spp., and infections by extracellular macroparasites, pinworms. For Eimeria, we found lower intensities in hybrid hosts than in parental mice but no evidence of lowered probability of infection or increased mortality in the centre of the hybrid zone. This means ecological factors are very unlikely to be responsible for the reduced load of infected hybrids. Focusing on parasite intensity (load in infected hosts), we also corroborated reduced pinworm loads reported for hybrid mice in previous studies. We conclude that intensity of diverse parasites, including the previously unstudied Eimeria, is reduced in hybrid mice compared to parental subspecies. We suggest caution in extrapolating this to differences in hybrid host fitness in the absence of, for example, evidence for a link between parasitemia and health.

Detection and quantification of house mouse Eimeria at the species level - Challenges and solutions for the assessment of coccidia in wildlife.

Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals. Parasite species - specific prevalences are especially important when studying evolutionary questions in wild populations. We tested whether increased host population density increases prevalence of individual Eimeria species at the farm level, as predicted by epidemiological theory. We studied free-living commensal populations of the house mouse (Mus musculus) in Germany, and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred niche of each parasite species along the distal-proximal axis of the intestine. Parasite genotyping from tissue samples provides additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.7%; 95% CI 13.2-20.7), E. falciformis (4.2%; 95% CI 2.6-6.8) and E. vermiformis (1.9%; 95% CI 0.9-3.8). We also find that mice in dense populations are more likely to be infected with E. falciformis and E. ferrisi. We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We show and discuss how such data can help to test hypotheses in ecology, evolution and epidemiology on a species level.

Maternal-embryonic metabolic and antioxidant response of Chapalichthys pardalis (Teleostei: Goodeidae) induced by exposure to 3,4-dichloroaniline.

Chapalichthys pardalis is a viviparous fish, microendemic to the Tocumbo Region in the state of Michoacán, Mexico. Despite the peculiar type of reproduction of goodeid fish and their mother-embryo interaction, the effects on embryos induced by maternal exposure to aquatic xenobiotics are still unknown. The objective of the present work was to determine the maternal-embryonic metabolic and antioxidant response of C. pardalis exposed to 3,4-dichloroaniline (3,4-DCA), a compound considered highly noxious to the environment because of its high toxicity and persistence, which has been used as reference toxicant in toxicological bioassays. We determined the median lethal concentration (LC50, 96 h) and then exposed pregnant females to 3.3, 2.5, and 0.5 mg L-1 of 3,4-DCA (equivalent to LC1, LC0.01, and LC50/10, respectively) during 21 days. We assessed the activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), macromolecules content (proteins, lipids, carbohydrates), glucose, and lactate concentration, as well as the oxidative damage, by measuring thiobarbituric acid reactive substances, and protein oxidation. To interpret results, we used the integrated biomarker response (IBRv2). The average LC50 was of 5.18 mg L-1 (4.8-5.5 mg L-1; p = 0.05). All females exposed to concentrations of 3.3 and 2.5 mg L-1 lost 100% of the embryos during the bioassay, whereas those exposed to 0.5 mg L-1 showed alterations in the antioxidant activity and oxidative damage, being the embryos and the maternal liver the most affected, with IBRv2 values of 10.09 and 9.21, respectively. Damage to macromolecules was greater in embryos and the maternal liver, with IBRv2 of 16.14 and 8.40, respectively. We conclude that exposure to xenobiotics, like 3,4-DCA, in species with a marked maternal-embryonic interaction represents a potential risk for the development and survival of the descendants, thereby, potentially affecting the future of the population.

Exposure to silver nanoparticles produces oxidative stress and affects macromolecular and metabolic biomarkers in the goodeid fish Chapalichthys pardalis.

Silver nanoparticles (AgNPs) are the most commercialized nanomaterial worldwide, mainly due to their microbicidal activity. Although, AgNPs have been shown to be toxic to aquatic species, their effect on endemic fish, like Goodeidae, has not been demonstrated. Endemic species are under strong pressures by anthropogenic contamination and destruction of their habitat; therefore, we studied adult Chapalichthys pardalis, an endemic fish of Mexico. We evaluated the toxic effect of AgNPs through oxidative stress, macromolecular and metabolic biomarkers. We determined the LC50 (96h) and performed subchronic tests (21days) using sublethal AgNPs concentrations (equivalent to CL1 and CL10). At the end of the bioassay, we quantified 10 stress biomarkers in the liver, gills, and muscle, including the antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione [GPx]), thiobarbituric acid reactive species (TBARS), protein oxidation (CO), macromolecules (proteins, lipids, and carbohydrates), and metabolites (glucose and lactate). In addition, we determined the integrated biomarkers response (IBR). LC50 was of 10.32mgL-1. Results of subchronic exposure (21days) revealed that AgNPs produce oxidative stress in C. pardalis adults, as evidenced by a diminution in antioxidant enzymes activity and an increase in TBARS and oxidized proteins. AgNPs also diminished levels of macromolecules and generated a high-energy consumption, reflected in the reduction of glucose levels, although lactate levels were not altered. The IBR analysis evidenced that the largest effect was produced in organisms exposed to LC10, being the liver and gills the organs with the greatest damage. Results demonstrated that exposure to AgNPs induces acute and chronic toxic effects on C. pardalis and forewarns about the impact that these nanomaterials can exert on these ecologically relevant aquatic organisms.

First molecular evidence of Hepatozoon canis in domestic dogs and ticks in fragmented rainforest areas in Mexico.

The tick-borne pathogens of the genus Hepatozoon affect domestic animals and wildlife; their prevalence has risen around the world in the past years. In Mexico there is not enough data available about their surveillance. This study aimed to detect the prevalence of Hepatozoon by PCR in domestic animals and ticks from a fragmented rainforest area from southeast Mexico and analyze the phylogeographic structure of the parasites detected. The total prevalence of H. canis in mammals was 9.7% (20/206; 95% Confidence limits: 6.0-14.6%), being dogs the species with the highest prevalence, of 63.3% (19/30; 95% Confidence limits: 43.9-80.1%). The phylogenetic analysis revealed that sequences from this study were closer to the sequence of H. canis of domestic origin, rather than from wild origin, but in an independent cluster. Haplotypes from our study were geographically restricted to Mexico and the closest haplotype was from Brazil. Ticks that resulted positive by PCR were identified as Amblyomma cajennense (A. mixtus) and Rhipicephalus turanicus. Under fragmented and disturbed conditions of habitat in Balancan, the presence of H. canis may represent a potential risk for other species of domestic and wildlife animals. To the knowledge of the authors, this study represents the first molecular finding of H. canis in Mexico in both domestic animals and ticks. This research lays the groundwork for further studies in order to elucidate the relationships between domestic hosts, wildlife and ticks and describe the life cycle of this parasite in the area.