Performance of the Vetscan Imagyst in point-of-care detection of Giardia duodenalis in canine fecal samples.

Giardia duodenalis is a common parasite of the gastrointestinal tract of dogs, with an especially high prevalence in dogs <1-y-old. Methods for detecting G. duodenalis are point-of-care (POC) tests such as lateral-flow tests or fecal flotation. The Vetscan Imagyst (Zoetis) is a new POC device for the detection of G. duodenalis in fecal samples using zinc sulfate flotation, automated slide scanning, and image recognition with artificial intelligence. Vetscan results are the number of Giardia cysts per coverslip. We compared the performance of the Vetscan and another POC test (SNAP Giardia test; Idexx) with a direct immunofluorescence assay (IFA) performed in a specialized parasitology laboratory as the reference test. We included 164 dogs <19-mo-old. We used pooled fecal samples from 3 defecations gained within 2-3 d and tested the repeatability of the Vetscan by triplicate measurement. Compared to IFA, Vetscan had a diagnostic sensitivity of 88.4% and specificity of 98.1%; SNAP had a diagnostic sensitivity of 74.4% and specificity of 98.1%. A variation coefficient of 67.0% was determined for the Vetscan results. The performance of the Vetscan is acceptable for the qualitative evaluation of fecal samples (Giardia positive or negative), and the device can be used by untrained personnel. Given its high variation coefficient, we do not recommend the Vetscan for monitoring the number of cysts.

Comparing treatment effects on dogs with acute hemorrhagic diarrhea syndrome: fecal microbiota transplantation, symptomatic therapy, or antibiotic treatment.

OBJECTIVE: Dogs with acute hemorrhagic diarrhea syndrome (AHDS) present with similar clinical signs and histopathological findings as dogs with parvovirosis, in which fecal microbiota transplantation (FMT) has led to a significantly faster resolution of diarrhea and shorter hospitalization times. We investigated whether FMT results in faster clinical improvement and normalization of the intestinal microbiome compared to standard treatment. ANIMALS: 32 client-owned dogs with AHDS. METHODS: A prospective, double-anonymized clinical trial included 3 groups: symptomatic treatment (n = 12), FMT treatment (FMTT; 12), and antibiotic treatment (AT; 8). Clinical improvement was determined on the basis of AHDS index, changes in the microbiome based on the dysbiosis index, and PCR results for clostridial strains. RESULTS: Overall, no significant differences in clinical scores between the treatment groups over time were detected except on day 2 (higher AHDS index in the AT group compared to FMTT group; P = .046). The dysbiosis index increased and P hiranonis decreased on day 1 in some dogs, but these changes were transient in the symptomatic treatment and FMTT groups. In the AT group, the dysbiosis index was persistently elevated and 4 of 8 dogs showed a reduced abundance of P hiranonis on day 42. In 67% of the dogs on day 1, NetF-encoding Clostridium perfringens was detected and enterotoxin-encoding strains increased, but these changes were transient in all dogs, regardless of therapy. CLINICAL RELEVANCE: Overall, in dogs with AHDS, neither FMT nor AT resulted in faster clinical improvement. In addition, C perfringens strains are self-limiting and do not require antibiotic therapy.

The Effect of Metronidazole versus a Synbiotic on Clinical Course and Core Intestinal Microbiota in Dogs with Acute Diarrhea.

The usefulness of antibiotics in dogs with acute diarrhea (AD) is controversial. It is also unclear what effect metronidazole has on potential enteropathogens such as Clostridium perfringens and Escherichia coli. Thus, the aim of this study was to evaluate the effect of metronidazole vs. a synbiotic on the clinical course and core intestinal bacteria of dogs with AD. Twenty-seven dogs with AD were enrolled in this prospective, randomized, blinded clinical trial and treated with either metronidazole (METg) or a synbiotic (SYNg; E. faecium DSM 10663; NCIMB 10415/4b170). The Canine Acute Diarrhea Severity (CADS) index was recorded daily for eleven days. Bacteria were quantified using qPCR. Data were analyzed using mixed models with repeated measures. A higher concentration of E. coli was observed in the METg group vs. the SYNg group on Day 6 (p < 0.0001) and Day 30 (p = 0.01). Metronidazole had no effect on C. perfringens. C. hiranonis was significantly lower in the METg group than in the SYNg group on Days 6 and 30 (p < 0.0001; p = 0.0015). No significant differences were observed in CADS index, fecal consistency, or defecation frequency between treatment groups (except for the CADS index on one single day). In conclusion, metronidazole negatively impacts the microbiome without affecting clinical outcomes. Thus, synbiotics might be a preferred treatment option for dogs with AD.

Evaluation of Intestinal Barrier Dysfunction with Serum Iohexol Concentration in Dogs with Acute Hemorrhagic Diarrhea Syndrome.

Histopathologic examination of intestinal biopsies from dogs with acute hemorrhagic diarrhea syndrome (AHDS) reveals necrotizing enteritis and epithelial integrity loss. Serum iohexol measurement has been utilized to assess intestinal permeability. Our hypothesis is that dogs with AHDS have increased intestinal permeability, which is associated with the severity of clinical signs. In this prospective case-control study, 53 client-owned dogs (28 AHDS, 25 healthy controls) were evaluated. Clinical severity was assessed using the AHDS index and systemic inflammatory response syndrome (SIRS) criteria. Simultaneously, dogs received oral iohexol, and serum iohexol concentrations (SICs) were measured two hours later. Results indicated significantly higher (p = 0.002) SIC in AHDS dogs (median: 51 µg/mL; min-max: 9-246) than in healthy controls (30 µg/mL; 11-57). There was a significant positive correlation between AHDS index and SIC (rS = 0.4; p = 0.03) and a significant negative between SIC and serum albumin concentrations (Pearson r = -0.55; p = 0.01). Dogs with severe AHDS (mean 106 µg/mL; range: 17-246) demonstrated significantly higher (p = 0.002) SIC than those with mild to moderate disease (29 µg/mL; 9-54). These findings underscore the association between intestinal permeability and clinical severity in dogs with AHDS assessed by iohexol.

[Feline and canine giardiosis: An Update]. / Ein Update zur felinen und caninen Giardiose.

Giardia duodenalis is a facultative pathogenic intestinal parasite. Giardiosis in dogs and cats may appear with or without clinical signs. Typical signs include diarrhea with or without vomiting. The prevalence in young animals is high and may amount to up to 50%. There are 8 different genotypes (A - H), which are called assemblages. Assemblages C and D are most common in dogs and assemblage F most frequent in cats. However, animals may also be infected with the zoonotically effective assemblages A and B or exhibit mixed infections. The immunofluorescence test (IFA), the enzyme-linked immunosorbent assay (ELISA) and fecal centrifugation using zinc sulphate solution are currently recommended as diagnostic methods. Polymerase chain reaction (PCR) may be used to determine the corresponding assemblage. Approved treatments for giardiosis include fenbendazole and metronidazole. In addition, undertaking specific hygiene measures is warranted. Only animals showing clinical signs or those living in the same household with high-risk patients (e. g. immunosuppressed humans) are recommended to receive medication. The aim of treatment is clinical improvement of the diseased dogs and cats. Frequently, complete elimination of Giardia is not attained.

Correlation between Targeted qPCR Assays and Untargeted DNA Shotgun Metagenomic Sequencing for Assessing the Fecal Microbiota in Dogs.

DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI < 0 with any targeted taxa outside the reference interval, RI), mild-moderate changes (R = 0.24, 0 < DI < 2), and significant dysbiosis (R = 0.54, 0.73, and 0.91 for DI > 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs.

Correction: Effect of probiotic treatment on the clinical course, intestinal microbiome, and toxigenic Clostridium perfringens in dogs with acute hemorrhagic diarrhea.

[This corrects the article DOI: 10.1371/journal.pone.0204691.].

Observing many researchers using the same data and hypothesis reveals a hidden universe of uncertainty.

This study explores how researchers' analytical choices affect the reliability of scientific findings. Most discussions of reliability problems in science focus on systematic biases. We broaden the lens to emphasize the idiosyncrasy of conscious and unconscious decisions that researchers make during data analysis. We coordinated 161 researchers in 73 research teams and observed their research decisions as they used the same data to independently test the same prominent social science hypothesis: that greater immigration reduces support for social policies among the public. In this typical case of social science research, research teams reported both widely diverging numerical findings and substantive conclusions despite identical start conditions. Researchers' expertise, prior beliefs, and expectations barely predict the wide variation in research outcomes. More than 95% of the total variance in numerical results remains unexplained even after qualitative coding of all identifiable decisions in each team's workflow. This reveals a universe of uncertainty that remains hidden when considering a single study in isolation. The idiosyncratic nature of how researchers' results and conclusions varied is a previously underappreciated explanation for why many scientific hypotheses remain contested. These results call for greater epistemic humility and clarity in reporting scientific findings.

Stability of a dominant sponge-symbiont in spite of antibiotic-induced microbiome disturbance.

Marine sponges are known for their complex and stable microbiomes. However, the lack of a gnotobiotic sponge-model and experimental methods to manipulate both the host and the microbial symbionts currently limit our mechanistic understanding of sponge-microbial symbioses. We have used the North Atlantic sponge species Halichondria panicea to evaluate the use of antibiotics to generate gnotobiotic sponges. We further asked whether the microbiome can be reestablished via recolonization with the natural microbiome. Experiments were performed in marine gnotobiotic facilities equipped with a custom-made, sterile, flow-through aquarium system. Bacterial abundance dynamics were monitored qualitatively and quantitatively by 16 S rRNA gene amplicon sequencing and qPCR, respectively. Antibiotics induced dysbiosis by favouring an increase of opportunistic, antibiotic-resistant bacteria, resulting in more complex, but less specific bacteria-bacteria interactions than in untreated sponges. The abundance of the dominant symbiont, Candidatus Halichondribacter symbioticus, remained overall unchanged, reflecting its obligately symbiotic nature. Recolonization with the natural microbiome could not reverse antibiotic-induced dysbiosis. However, single bacterial taxa that were transferred, successfully recolonized the sponge and affected bacteria-bacteria interactions. By experimentally manipulating microbiome composition, we could show the stability of a sponge-symbiont clade despite microbiome dysbiosis. This study contributes to understanding both host-bacteria and bacteria-bacteria interactions in the sponge holobiont.

Biodiversity, environmental drivers, and sustainability of the global deep-sea sponge microbiome.

In the deep ocean symbioses between microbes and invertebrates are emerging as key drivers of ecosystem health and services. We present a large-scale analysis of microbial diversity in deep-sea sponges (Porifera) from scales of sponge individuals to ocean basins, covering 52 locations, 1077 host individuals translating into 169 sponge species (including understudied glass sponges), and 469 reference samples, collected anew during 21 ship-based expeditions. We demonstrate the impacts of the sponge microbial abundance status, geographic distance, sponge phylogeny, and the physical-biogeochemical environment as drivers of microbiome composition, in descending order of relevance. Our study further discloses that fundamental concepts of sponge microbiology apply robustly to sponges from the deep-sea across distances of >10,000 km. Deep-sea sponge microbiomes are less complex, yet more heterogeneous, than their shallow-water counterparts. Our analysis underscores the uniqueness of each deep-sea sponge ground based on which we provide critical knowledge for conservation of these vulnerable ecosystems.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Frequency of signs of chronic gastrointestinal disease in dogs after an episode of acute hemorrhagic diarrhea.

BACKGROUND: Acute enteropathy is a trigger of chronic gastrointestinal (GI) disease in humans. OBJECTIVE: To report the prevalence of and explore possible risk factors for signs of chronic GI disease in dogs after an episode of acute hemorrhagic diarrhea (AHD). ANIMALS: One hundred and fifty-one dogs, 80 dogs with a historical diagnosis of AHD, 71 control dogs with no history of AHD. METHODS: In this retrospective longitudinal study, data were collected from dogs with a historical diagnosis of AHD and healthy controls matched by breed, age and sex, aged between 1 year and 15 years of age, for which a follow-up of at least 12 months after enrolment was available. Dog owners responded to a questionnaire to determine the history of signs of chronic GI disease. RESULTS: There was a higher prevalence of signs of chronic GI disease in the dogs with a previous episode of AHD compared to control dogs (AHD 28%; controls 13%; P = .03; odds ratio = 2.57; confidence interval [CI] 95% 1.12-6.31) over a similar observation time (median 4 years; range, 1-12 years). CONCLUSIONS AND CLINICAL IMPORTANCE: Severe intestinal mucosal damage and associated barrier dysfunction might trigger chronic GI disease later in life.

Biogeography rather than substrate type determines bacterial colonization dynamics of marine plastics.

Since the middle of the 20th century, plastics have been incorporated into our everyday lives at an exponential rate. In recent years, the negative impacts of plastics, especially as environmental pollutants, have become evident. Marine plastic debris represents a relatively new and increasingly abundant substrate for colonization by microbial organisms, although the full functional potential of these organisms is yet to be uncovered. In the present study, we investigated plastic type and incubation location as drivers of marine bacterial community structure development on plastics, i.e., the Plastisphere, via 16S rRNA amplicon analysis. Four distinct plastic types: high-density polyethylene (HDPE), linear low-density polyethylene (LDPE), polyamide (PA), polymethyl methacrylate (PMMA), and glass-slide controls were incubated for five weeks in the coastal waters of four different biogeographic locations (Cape Verde, Chile, Japan, South Africa) during July and August of 2019. The primary driver of the coastal Plastisphere composition was identified as incubation location, i.e., biogeography, while substrate type did not have a significant effect on bacterial community composition. The bacterial communities were consistently dominated by the classes Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia, irrespective of sampling location or substrate type, however a core bacterial Plastisphere community was not observable at lower taxonomic levels. Overall, this study sheds light on the question of whether bacterial communities on plastic debris are shaped by the physicochemical properties of the substrate they grow on or by the marine environment in which the plastics are immersed. This study enhances the current understanding of biogeographic variability in the Plastisphere by including biofilms from plastics incubated in the previously uncharted Southern Hemisphere.

Marine sponges maintain stable bacterial communities between reef sites with different coral to algae cover ratios.

Marine sponges play a major ecological role in recycling resources on coral reef ecosystems. The cycling of resources may largely depend on the stability of the host-microbiome interactions and their susceptibility to altered environmental conditions. Given the current coral to algal phase shift on coral reefs, we investigated whether the sponge-associated bacterial communities of four sponge species, with either high or low microbial abundances (HMA and LMA), remain stable at two reefs sites with different coral to algae cover ratios. Additionally, we assessed the bacterial community composition of two of these sponge species before and after a reciprocal transplantation experiment between the sites. An overall stable bacterial community composition was maintained across the two sites in all sponge species, with a high degree of host-specificity. Furthermore, the core bacterial communities of the sponges remained stable also after a 21-day transplantation period, although a minor shift was observed in less abundant taxa (< 1%). Our findings support the conclusion that host identity and HMA-LMA status are stronger traits in shaping bacterial community composition than habitat. Nevertheless, long-term microbial monitoring of sponges along with benthic biomass and water quality assessments are needed for identifying ecosystem tolerance ranges and tipping points in ongoing coral reef phase shifts.

DNA-stable isotope probing (DNA-SIP) identifies marine sponge-associated bacteria actively utilizing dissolved organic matter (DOM).

Sponges possess exceptionally diverse associated microbial communities and play a major role in (re)cycling of dissolved organic matter (DOM) in marine ecosystems. Linking sponge-associated community structure with DOM utilization is essential to understand host-microbe interactions in the uptake, processing, and exchange of resources. We coupled, for the first time, DNA-stable isotope probing (DNA-SIP) with 16S rRNA amplicon sequencing in a sponge holobiont to identify which symbiotic bacterial taxa are metabolically active in DOM uptake. Parallel incubation experiments with the sponge Plakortis angulospiculatus were amended with equimolar quantities of unlabelled (12 C) and labelled (13 C) DOM. Seven bacterial amplicon sequence variants (ASVs), belonging to the phyla PAUC34f, Proteobacteria, Poribacteria, Nitrospirae, and Chloroflexi, were identified as the first active consumers of DOM. Our results support the predictions that PAUC34f, Poribacteria, and Chloroflexi are capable of organic matter degradation through heterotrophic carbon metabolism, while Nitrospirae may have a potential mixotrophic metabolism. We present a new analytical application of DNA-SIP to detect substrate incorporation into a marine holobiont with a complex associated bacterial community and provide new experimental evidence that links the identity of diverse sponge-associated bacteria to the consumption of DOM.

Acute Hemorrhagic Diarrhea Syndrome in Dogs.

Acute hemorrhagic diarrhea syndrome is defined as sudden onset of severe bloody diarrhea frequently associated with vomiting, which results in severe, sometimes life-threatening dehydration. Although there is strong evidence that clostridial overgrowth and toxin release is responsible for the pathogenesis of the disease, the diagnosis is still based on exclusion of other causes for acute hemorrhagic diarrhea. With early and appropriate treatment, mainly based on fluid therapy, the prognosis is good and complications such as sepsis or severe hypoalbuminemia rarely occur.

Comparative Microbiome and Metabolome Analyses of the Marine Tunicate Ciona intestinalis from Native and Invaded Habitats.

Massive fouling by the invasive ascidian Ciona intestinalis in Prince Edward Island (PEI, Canada) has been causing devastating losses to the local blue mussel farms. In order to gain first insights into so far unexplored factors that may contribute to the invasiveness of C. intestinalis in PEI, we undertook comparative microbiome and metabolome studies on specific tissues from C. intestinalis populations collected in invaded (PEI) and native regions (Helgoland and Kiel, Germany). Microbial community analyses and untargeted metabolomics revealed clear location- and tissue-specific patterns showing that biogeography and the sampled tissue shape the microbiome and metabolome of C. intestinalis. Moreover, we observed higher microbial and chemical diversity in C. intestinalis from PEI than in the native populations. Bacterial OTUs specific to C. intestinalis from PEI included Cyanobacteria (e.g., Leptolyngbya sp.) and Rhodobacteraceae (e.g., Roseobacter sp.), while populations from native sampling sites showed higher abundances of e.g., Firmicutes (Helgoland) and Epsilonproteobacteria (Kiel). Altogether 121 abundant metabolites were putatively annotated in the global ascidian metabolome, of which 18 were only detected in the invasive PEI population (e.g., polyketides and terpenoids), while six (e.g., sphingolipids) or none were exclusive to the native specimens from Helgoland and Kiel, respectively. Some identified bacteria and metabolites reportedly possess bioactive properties (e.g., antifouling and antibiotic) that may contribute to the overall fitness of C. intestinalis. Hence, this first study provides a basis for future studies on factors underlying the global invasiveness of Ciona species.

On the way to specificity - Microbiome reflects sponge genetic cluster primarily in highly structured populations.

Most animals, including sponges (Porifera), have species-specific microbiomes. Which genetic or environmental factors play major roles structuring the microbial community at the intraspecific level in sponges is, however, largely unknown. In this study, we tested whether geographic location or genetic structure of conspecific sponges influences their microbial assembly. For that, we used three sponge species with different rates of gene flow, and collected samples along their entire distribution range (two from the Mediterranean and one from the Southern Ocean) yielding a total of 393 samples. These three sponge species have been previously analysed by microsatellites or single nucleotide polymorphisms, and here we investigate their microbiomes by amplicon sequencing of the microbial 16S rRNA gene. The sponge Petrosia ficiformis, with highly isolated populations (low gene flow), showed a stronger influence of the host genetic distance on the microbial composition than the spatial distance. Host-specificity was therefore detected at the genotypic level, with individuals belonging to the same host genetic cluster harbouring more similar microbiomes than distant ones. On the contrary, the microbiome of Ircinia fasciculata and Dendrilla antarctica - both with weak population structure (high gene flow) - seemed influenced by location rather than by host genetic distance. Our results suggest that in sponge species with high population structure, the host genetic cluster influence the microbial community more than the geographic location.

Microbial Strategies for Survival in the Glass Sponge Vazella pourtalesii.

Few studies have explored the microbiomes of glass sponges (Hexactinellida). The present study seeks to elucidate the composition of the microbiota associated with the glass sponge Vazella pourtalesii and the functional strategies of the main symbionts. We combined microscopic approaches with metagenome-guided microbial genome reconstruction and amplicon community profiling toward this goal. Microscopic imaging revealed that the host and microbial cells appeared within dense biomass patches that are presumably syncytial tissue aggregates. Based on abundances in amplicon libraries and metagenomic data, SAR324 bacteria, Crenarchaeota, Patescibacteria, and Nanoarchaeota were identified as abundant members of the V. pourtalesii microbiome; thus, their genomic potentials were analyzed in detail. A general pattern emerged in that the V. pourtalesii symbionts had very small genome sizes, in the range of 0.5 to 2.2 Mb, and low GC contents, even below those of seawater relatives. Based on functional analyses of metagenome-assembled genomes (MAGs), we propose two major microbial strategies: the "givers," namely, Crenarchaeota and SAR324, heterotrophs and facultative anaerobes, produce and partly secrete all required amino acids and vitamins. The "takers," Nanoarchaeota and Patescibacteria, are anaerobes with reduced genomes that tap into the microbial community for resources, e.g., lipids and DNA, likely using pilus-like structures. We posit that the existence of microbial cells in sponge syncytia together with the low-oxygen conditions in the seawater environment are factors that shape the unique compositional and functional properties of the microbial community associated with V. pourtalesii IMPORTANCE We investigated the microbial community of V. pourtalesii that forms globally unique, monospecific sponge grounds under low-oxygen conditions on the Scotian Shelf, where it plays a key role in its vulnerable ecosystem. The microbial community was found to be concentrated within biomass patches and is dominated by small cells (<1 µm). MAG analyses showed consistently small genome sizes and low GC contents, which is unusual compared to known sponge symbionts. These properties, as well as the (facultatively) anaerobic metabolism and a high degree of interdependence between the dominant symbionts regarding amino acid and vitamin synthesis, are likely adaptations to the unique conditions within the syncytial tissue of their hexactinellid host and the low-oxygen environment.