Fresh Feces Research Articles

Comparison of commonly used software pipelines for analyzing fungal metabarcoding data.

Metabarcoding targeting the internal transcribed spacer (ITS) region is commonly used to characterize fungal communities of various environments. Given their size and complexity, raw ITS sequences are necessarily processed and quality-filtered with bioinformatic pipelines. However, such pipelines are not yet standardized, especially for fungal communities, and those available may produce contrasting results. While some pipelines cluster sequences based on a specified percentage of base pair similarity into operational taxonomic units (OTUs), others utilize denoising techniques to infer amplicon sequencing variants (ASVs). While ASVs are now considered a more accurate representation of taxonomic diversity for prokaryote communities based on 16S rRNA amplicon sequencing, the applicability of this method for fungal ITS sequences is still debated. Here we compared the performance of two commonly used pipelines DADA2 (inferring ASVs) and mothur (clustering OTUs) on fungal metabarcoding sequences originating from two different environmental sample types (fresh bovine feces and pasture soil). At a 99% OTU similarity threshold, mothur consistently identified a higher fungal richness compared to DADA2. In addition, mothur generated homogenous relative abundances across multiple technical replicates (n = 18), while DADA2 results for the same replicates were highly heterogeneous. Our study highlights a potential pipeline-associated bias in fungal metabarcoding data analysis of environmental samples. Based on the homogeneity of relative abundances across replicates and the capacity to detect OTUs/ASVs, we suggest using OTU clustering with a similarity of 97% as the most appropriate option for processing fungal metabarcoding data.

Freeze-dried fecal microorganisms as an effective biomaterial for the treatment of calves suffering from diarrhea.

Fecal microbiota transplantation (FMT) is a therapeutic modality for treating neonatal calf diarrhea. Several practical barriers, including donor selection, fecal collection, and a limited timeframe for FMT, are the main constraints to using fresh feces for implementing on-farm FMT. We report the utility of FMT with pretreated ready-to-use frozen (F) or freeze-dried (FD) microorganisms for treating calf diarrhea. In total, 19 FMT (F-FMT, n = 10 and FD-FMT, n = 9) treatments were conducted. Both FMT treatments were 100% clinically effective; however, multi-omics analysis showed that FD-FMT was superior to F-FMT. Machine learning analysis with SourceTracker confirmed that donor microbiota was retained four times better in the recipient calves treated with FD-FMT than F-FMT. A predictive model based on receiver operating characteristic curve analysis and area under the curve showed that FD-FMT was more discriminative than F-FMT of the observed changes in microbiota and metabolites during disease recovery. These results provide new insights into establishing methods for preparing fecal microorganisms to increase the quality of FMT in animals and may contribute to FMT in humans.

Fiber composition, in vitro true digestibility, and fiber degradation of hempseed meal compared to mainstream feedstuffs using equine feces as microbial inoculum within the DaisyII Incubator.

Recent changes in legislation have put hempseed meal (Cannabis sativa L.; HSM) under consideration as a potential livestock feedstuff. Digestibility of HSM has been researched in other species, however, there are little data in horses. The study objective was to determine the in vitro true digestibility (IVTD) of HSM compared to common equine feedstuffs. An in vitro split-split plot design used four geldings (8.5 ± 3.9yr; 558.4 ± 31.0kg; 6.0 ± 0.4 BCS) to investigate the IVTD of HSM compared to alfalfa hay (ALF), Coastal Bermudagrass hay (CBH), soybean meal (SBM), rice bran pellets (RBP), and a commercial concentrate (Bluebonnet Equilene Pellets; EQU). Fresh feces were collected after a 21-d acclimation period and used as incubation inoculum. Dried ground feed samples were incubated in ANKOM filter bags within 2 DaisyII incubators for 48h. Split samples were analyzed for pre-fermentation neutral detergent fiber (NDF). Incubated filter bags were dried and analyzed for NDF to determine IVTD and fiber degradation. Hempseed meal NDF was greater than ALF (P<0.001), RBP (P<0.001), and SBM (P<0.001) but less than CBH (P<0.001) and tended to be greater than EQU (P=0.077). The IVTD of HSM was greater than CBH (P<0.001) but less than all other feedstuffs (P<0.001). The results indicate that HSM has an NDF and IVTD between ALF and CBH. Due to its lower digestibility and NDF content compared to concentrates, HSM has potential to be used as a fiber source in equine rations.

Whole Mitochondrial DNA Sequencing Using Fecal Samples from Domestic Dogs

Medical care for domestic dogs is now respected worldwide as being at a similar level to that of humans. We previously established a test method to determine whole mitochondrial DNA (mtDNA) using oral mucosal DNA that may be useful for medical care and welfare. However, the sample types tested in dogs are not limited to those obtained from the oral mucosa. Therefore, in the present study, we attempted to establish a test method to determine whole mtDNA sequences using feces, which represents the least invasive specimen. Two Japanese domestic dogs were used in the present study. DNA was extracted from approximately 100 mg of fresh feces from each dog, and PCRs were performed using four primer pairs that can amplify whole mtDNA. Following PCR, amplicons were pooled to create a DNA library using an experimental robot with an original program. Data were then acquired via NGS and data analysis was performed. The results showed that the whole mtDNA sequence of the two dogs was determined with high accuracy. Our results suggest that feces can be adapted for mitochondrial disease and individual identification testing and could serve as a useful testing method for the future medical care and welfare of domestic dogs.

293 Impact of black soldier fly larvae meal in a dry extruded food on nutritional quality and health in adult cats

Abstract Insects like the black soldier fly larvae (Hermetia illucens; BSFL) have been proposed as protein-rich feed ingredients to support the growing demand for meat. The BSFL hold also promise for use as a protein source in pet foods and, as such, it has been studied for palatability, nutritional quality and health effects in dogs and cats. This study aimed to evaluate the effects of including BSFL meal in a dry, extruded food on nutritional quality and health in healthy adult cats. Two similar compositions of dry, extruded foods were formulated to meet the FEDIAF nutrition guidelines for adult cats of which one was chicken meal-based (control, with 34.3% chicken meal) and the other BSFL meal-based (inclusion of 37.5% BSFL meal). BSFL meal contained 95.0% dry matter, 53.1% crude protein, 14.3% crude fat, 6.0% ash (Protix, Dongen, The Netherlands). Two groups of four cats were each fed one of the two foods for two 28-d periods in a crossover design, with sample collection during the last 7 d of each period. Cats were housed in groups of the same sex during the first 21 d of each period. Both foods were well-accepted by the cats. No health problems (including coat and skin condition) were noted throughout the study. Food type did not impact body weight (P = 0.850). Food intake was greater when cats were fed the BSFL-based food (P &amp;lt; 0.001). Consistency scores of fresh feces were optimal and did not differ between the foods. Compared with the control food, the food with BSFL meal had decreased apparent fecal digestibility values for dry matter, organic matter, nitrogen, and gross energy (P &amp;lt; 0.05) although all values were high. Digestibility values for acid-hydrolyzed fat were similar for both foods (P = 0.628). Diets resulted in changes in fermentation product concentrations, like fecal ammonia was less when cats fed this food compared with the control food (P &amp;lt; 0.001), but the fecal concentrations of the short-chain fatty acids and biogenic amines were generally increased (P &amp;lt; 0.05). Moreover, reduced fecal bacterial diversity and affected bacterial relative abundance suggested that inclusion of BSFL meal steered the microbial composition in the distal gut of cats. It was observed increased relative abundance of bacteria genus considered beneficial for host health like Bifidobacterium (P &amp;lt; 0.001) and Megasphaera (P &amp;lt; 0.036); and inhibition of genus Negativibacillus and Lachnoclostridium (P &amp;lt; 0.001), previously associated to digestive issues and negative for gut health. Overall, the data gathered in this study suggests that BSFL meal is an adequate alternative ingredient for extruded dry cat foods and may exert a prebiotic effect on intestinal microbiota.

Characterization of the gut butyrate-producing bacteria and lipid metabolism in African green monkey as a natural host of simian immunodeficiency virus infection.

Natural hosts of simian immunodeficiency virus (SIV), such as the African green monkey (AGM), possess the ability to avoid acquired immune deficiency syndrome (AIDS) despite lifelong infection. The underlying mechanisms are not completely understood. This study aimed to characterize the gut microbiome and metabolite profiles of different nonhuman primates (NHPs) to provide potential insight into AIDS resistance. Fresh feces from Cynomolgus macaques (CMs), and Rhesus macaques (RMs), SIV- AGMs (AGM_N), and SIV+ AGMs (AGM_P) were collected and used for metagenomic sequencing and metabonomic analysis. Compared with CMs and RMs, significant decreases in the abundances of Streptococcus , Alistipes , Treponema , Bacteroides , and Methanobrevibacter ( P < 0.01), and significant increases in the abundances of Clostridium , Eubacterium , Blautia , Roseburia , Faecalibacterium , and Dialister ( P < 0.01) were detected in AGM_N. Compared with AGM_N, a trend toward increased abundances of Streptococcus and Roseburia were found in AGM_P. The levels of metabolites involved in lipid metabolism and butanoate metabolism significantly differed among AGM_P, AGM_N and CM ( P < 0.05). Our data, for the first time, demonstrated distinguishing features in the abundances of butyrate-producing bacteria and lipid metabolism capacities between different NHP hosts of SIV infection. These findings may correlate with the different characteristics observed among these hosts in the maintenance of intestinal epithelial barrier integrity, regulation of inflammation, and provide insights into AIDS resistance in AGMs.

Proposal of Lactobacillus amylovorus subsp. animalis subsp. nov. and an emended description of Lactobacillus amylovorus.

Seven novel lactic acid bacterial strains (BF125T, BF186, TKL145, YK3, YK6, YK10 and NSK) were isolated from the fresh faeces of Japanese black beef cattle and weanling piglets, spent mushroom substrates, or steeping water of a corn starch production plant. These strains are rod-shaped, Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, cytochrome oxidase-negative, facultatively anaerobic, and homofermentative. Strain BF125T did not produce any gas from glucose; both d- and l-lactate were produced as end-products of glucose (D/L, 40 : 60). Growth occurred at 30-45 °C (optimum, 37 °C), pH 5.0-8.0 (optimum, pH 6.0), and with NaCl concentration of 1.0-3.0% (w/v). The G+C content of genomic DNA of strain BF125T was 37.8 mol% (whole-genome analysis). The major fatty acids were C16 : 0, C18 : 1 ω9c, C19 cyclopropane 9, 10, and summed feature 10. The 16S rRNA gene in strain BF125T showed high similarity to that of the type strain of Lactobacillus amylovorus (99.93%), and the other isolates were also identified as L. amylovorus based on these similarities. A phylogenetic tree based on the core genomes of L. amylovorus strains (n=54), including the seven isolates, showed that they could be divided into two clusters. Strains YK3, YK6, YK10, and NSK were in the first cluster, along with the type strain DSM 20531T, while the second cluster included isolates BF125T, BF186, TKL145, and other strains isolated from various animal origins. Phenotypic differences in fermentability were observed for lactose, salicin, and gentiobiose between these two groups. The intergroup digital DNA-DNA hybridization values (72.9-78.6%) and intergroup average nucleotide identity values (95.64-96.92%) were comparable to values calculated using datasets of other valid subspecies of the genus (ex-) Lactobacillus. In light of the physiological, genotypic, and phylogenetic evidence, we propose a novel subspecies of L. amylovorus, named Lactobacillus amylovorus subsp. animalis subsp. nov. (type strain BF125T=MAFF 212522T=DSM 115528T). Our findings also led to the automatic creation of Lactobacillus amylovorus subsp. amylovorus subsp. nov. and an emended description of the species L. amylovorus.

Cucurbitacin IIb alleviates colitis via regulating gut microbial composition and metabolites

Cucurbitacin IIb, a member of the triterpenoid family, exerts beneficial effects on intestinal diseases, including enteritis and bacillary dysentery. However, its effects and mechanisms of action on colitis have not yet been explored. In this study, we used a mouse model of dextran sulfate sodium (DSS)-induced colitis and explored the effects of cucurbitacin IIb on colitis symptoms, inflammatory responses, microbiota, and metabolite profiles. The results showed that cucurbitacin IIb alleviated colitis symptoms including body weight loss, an increase in the disease activity index, and elevated levels of myeloperoxidase and eosinophil peroxidase content. Additionally, it ameliorated intestinal morphology impairment, reduced the phosphorylation of NFκB protein, and mitigated accumulation of pro-inflammatory cytokines IL-6 and IL-1β. Furthermore, cucurbitacin IIb alleviated alterations in gut microbial composition and metabolites in DSS-treated mice. However, antibiotic treatment diminishes the beneficial effects of cucurbitacin IIb on colitis. We further found that transplantation of fresh feces or heat-inactivated feces from mice treated with cucurbitacin IIb to DSS-treated mice alleviated colitis, similar to the effects of cucurbitacin IIb. Collectively, our results suggest that cucurbitacin IIb exerted anti-inflammatory effects in colitis by regulating the microbiota composition and metabolites, thereby alleviating colitis symptoms.

Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.

To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated. 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method. There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.

Zoonotic potential of urban wildlife faeces, assessed through metabarcoding

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.

Astragalus Polysaccharide improves immunogenicity of influenza vaccine as well as modulate gut microbiota in BALB/c mice

BackgroundVaccination is the best way to prevent influenza virus infection, and insufficient antibodies make it difficult to resist influenza virus invasion. Astragalus Polysaccharide (APS) has a boosting effect on immunity, so we evaluate the effect of APS as an immune adjuvant for H1N1 influenza vaccines in this study. MethodsThe mice were immunized twice with influenza A (H1N1) vaccine and APS. Subsequently, the serum antibody levels were assessed using enzyme-linked immunosorbent assay (ELISA). The frequency of peripheral immune T cells was determined by flow cytometry. Following this, the immunized mice were exposed to a lethal dose of the virus, and changes in body weight and survival rates were recorded. Hematoxylin-eosin staining was employed to observe pathological alterations in lung and intestinal tissues. Western blot analysis was conducted to detect the expression of intestinal barrier function proteins (Occludin and Claudin-1). ELISA was utilized to measure the expression level of serum inflammatory cytokine TNF-α. Fresh mouse feces were collected after the initial immunization as well as after viral infection for 16S rRNA analysis aimed at detecting alterations in gut microbiota. ResultsCompared to the Hemagglutinin (HA) group, the APS group demonstrated higher levels of immunoglobulin G (IgG), IgG1, and IgG3, as well as neutralizing antibody levels. Additionally, it increased the frequency of CD8+ cells to enhance resistance against lethal infection. On day 14 post-infection, the high-dose APS group exhibited a higher survival rate (71.40 %) compared to the HA group (14.28 %), along with faster weight recovery. Furthermore, APS was found to ameliorate alveolar damage in lung tissue and rectify intestinal structural disorder. It also upregulated the expression levels of tight junction proteins Occludin and Claudin-1 in intestinal tissue while reducing serum TNF-α expression levels. In addition, populations of Colidextribacter, Peptococcaceae, and Ruminococcaceae were the dominant gut microbiota in the APS group after viral infection. ConclusionAPS has an immune-enhancing effect and is expected to be a novel adjuvant in the H1N1 influenza vaccine.

Molecular detection and genotyping of Toxoplasma gondii in stray cat feces from Khorramabad, West Iran

Cats, being the definitive host of Toxoplasma gondii, have a significant impact on the spread and outbreaks of the parasite. An essential factor in comprehending the transmission pattern of this parasite is an analysis of the genetic diversity distribution in cats infected with T. gondii. This study was aimed at determining the prevalence rate and genotyping of T. gondii in stray cat feces from Khorramabad, West Iran. In the years 2016–2017, 200 cats were sampled to get fresh feces specimens. Parasitological methods were utilized for the identification of oocysts. The DNA was isolated from the feces using a commercially available Genomic Mini Kit. In order to identify the genetic composition of T. gondii, we employed PCR-RFLP, sequencing, and phylogenetic analysis of the GRA6 target gene. No one of the samples tested positive for parasitology techniques. A total of 6.5 % (13/200) samples were positive when using the GRA6-PCR method. Based on PCR-RFLP results, all 13 samples were of T. gondii type III genotype. The nucleotide sequences of two samples from this study were found to be 5 % different from those of 12 references of T. gondii and one strain of Hammondia hamondi that was used as an external control. Based on the findings, molecular tests are more sensitive than parasitological methods. The RFLP approach revealed that type III of T. gondii is the prevailing and important genotype in Khorramabad, West Iran.

Single and Mixed Strains of Probiotics Reduced Hepatic Fat Accumulation and Inflammation and Altered Gut Microbiome in a Nonalcoholic Steatohepatitis Rat Model.

As gut dysbiosis has been implicated in the pathogenesis of nonalcoholic steatohepatitis (NASH), probiotic supplementation might be a potential treatment for this condition. The aim of this study was to evaluate the effects of single- and mixed-strain probiotics on the severity of NASH induced by a high-fat, high-fructose (HFHF) diet and their mechanisms of action. Male Sprague-Dawley rats were divided into four groups (n = 7 per group): control group, NASH group, NASH + single-strain group, and NASH + mixed-strain group. In the single-strain and mixed-strain groups, rats received Lactobacillus plantarum B7 and Lactobacillus rhamnosus L34 + Lactobacillus paracasei B13 by oral gavage once daily, respectively. The duration of the study was 6 weeks. Liver tissue was used for histopathology, hepatic fat content was assessed by Oil Red O staining and hepatic free fatty acid (FFA), and hepatic TLR4 and CD14 expression were assessed by immunohistochemistry. Fresh feces was collected for gut microbiota analysis. Liver histology revealed a higher degree of fat accumulation, hepatocyte ballooning, and lobular inflammation in the NASH group, which improved in probiotics-treated groups. The amounts of hepatic fat droplets and hepatic FFA levels were more pronounced in the NASH group than in the control and treatment groups. Serum TNF- α levels were significantly higher in the NASH group than in control and probiotic groups. The expression of CD14 and TLR4 increased in the NASH group as compared with the control and probiotics-treated groups. Alpha diversity was reduced in the NASH group, but increased in both treatment groups. The relative abundance of Lactobacillus significantly decreased in the NASH group, but increased in both treatment groups. The relative abundance of Akkermansia significantly increased in the NASH group, but decreased in both treatment groups. In conclusion, both single-strain and mixed-strain probiotics could improve NASH histology by suppressing inflammatory responses in the liver, with this improvement potentially being associated with changes in the gut microbiota.

Effects of Heat-Treated Bifidobacterium longum CECT-7384 Combined with Fibersol-2 on the Intestinal Health of Cats Submitted to an Abrupt Dietary Change: A Randomized Controlled Study.

Abrupt dietary change can disrupt the intestinal balance in felines. This study aimed to assess the impact of heat-treated Bifidobacterium longum CECT-7384 combined with Fibersol-2 on the intestinal health of adult cats before and after dietary change. We selected 24 British shorthair cats, dividing them into two groups. From day 1 to day 14, the control group received a lower protein (33%) concentration (LPF) diet, while the treated group received the same LPF diet supplemented with 0.16% functional additives, consisting of Bifidobacterium longum CECT-7384 combined with Fibersol-2. Subsequently, from day 15 to day 28, the control group transitioned to a higher protein (40%) concentration (HPF) diet, while the treated group received the same HPF diet supplemented with 0.16% functional additives. Blood and fresh feces were collected on day 0, 14, 17, 21, and 28 of the experiment. The results suggest that the use of heat-treated Bifidobacterium longum CECT-7384 combined with Fibersol-2 may improve gastrointestinal function in cats by reducing serum LPS levels and fecal pH, while increasing fecal sIgA levels. In addition, the functional additive regulates the fecal microbiota and its function, promoting intestinal homeostasis and colonization with beneficial bacteria such as Blautia. Furthermore, on day 28, there was a significant difference in fecal microbiota beta diversity between the two groups. In summary, the addition of heat-treated Bifidobacterium longum CECT-7384 combined with Fibersol-2 contributes to improving the intestinal health of adult cats affected by abrupt dietary change.

Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses

BackgroundCurrently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region.ResultsThe largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities.ConclusionsFrozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.

Molecular prevalence, subtype distribution, and zoonotic potential of Blastocystis sp. in wild rodents and shrews inhabiting Zhejiang province of China.

Globally, rodents and shrew populations constitute crucial elements of diverse environments and animal communities. It is imperative to study their population dynamics to mitigate any potential negative impact on humans, as they can be involved in the transmission of critical zoonotic agents, such as Blastocystis. Therefore, this study aimed to identify the prevalence and genetic composition of Blastocystis in wild rodents and shrews residing in the Zhejiang provinces of China. A total of 652 wild rodents and and shrews were captured from three different regions in Zhejiang Province from April 1st to October 31, 2023. The DNA was isolated by collecting fresh feces from the intestines of each rodent or and shrew. Rodent and shrew species were examined by vertebrate cytochrome b (cytb) analysis and PCR amplification. Blastocystis was also found in all fecal samples using PCR analysis and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. Among all the samples, 6.6% (43/652) showed a positive result for Blastocystis. In the results, 6 species of rodent and shrew were identified with Blastocystis, including Apodemus agrarius (n = 36) (2.8%), Niviventer confucianus (n = 75) (17.3%), Rattus losea (n = 18) (5.6%), R. norvegicus (n = 155) (2.6%), R. tanezumi (n = 86) (3.5%), and Suncus murinus (n = 282) (7.4%). The existence of 6 Blastocystis subtypes, ST4 (n = 33), ST1 (4), ST7 (n = 3), ST2 (n = 1), ST3 (n = 1), and ST5 (n = 1), were confirmed by sequence analysis. Based on the molecular data obtained, the wild rodents and shrews under investigation were found to be concurrently infected with zoonotic subtypes of Blastocystis, including ST1 to ST5 and ST7. This suggests that these animals could potentially pose a zoonotic threat to humans and other animals susceptible to Blastocystis infection.

Environmental effects on faecal genotyping success in mesocarnivores

AbstractNon-invasive genetic sampling can be used in research, monitoring, and conservation of wild animals to, for example, provide insights into diets, identify individuals and estimate population sizes. Non-invasive genetic sampling may be especially useful to monitor elusive species because DNA can be derived from materials such as hairs and faeces without handling individuals. However, the reliability of the results derived from this technique is dependent on the quality of DNA obtained from samples, which can deteriorate from exposure to environmental conditions and sample age. While freshness of the sample is an important factor, the combined effect of different field conditions on the genotyping success is not fully understood. To address this gap, we systematically investigated the effects of sample age and environmental conditions on genotyping success of faeces, in an experimental setting of four treatments that combined rain and temperature conditions typical of central European climates. We compared the performance of a microsatellite marker set and a SNP panel for red fox (Vulpes vulpes), as well as a microsatellite marker set for pine marten (Martes martes) from faeces resampled over 21 days. We found that genotyping success decreased significantly with sample age, however environmental treatments did not impact the success. Furthermore, the SNPs we used amplified more successfully over time than the respective microsatellites. Therefore, the use of SNPs instead of conventional microsatellites, when using faecal samples for analysis relying on correct amplifications, could be advantageous. We recommend to prioritise the collection of fresh faeces regardless of environmental conditions.

Effect of Dietary Addition of Fennel (Foeniculum vulgare) Seed on Growth Performance, Haemato-Biochemical Profile and Faecal Microbiota of Kankrej Calves

The experiment was conducted during the month of February and March, 2023 at Livestock Research Station, Kamdhenu University, Sardarkrushinagar, Gujarat, India to evaluate the effect of dietary addition of fennel (Foeniculum vulgare) seed on growth performance, haemato-biochemical profile and faecal microbiota of Kankrej calves. Eighteen Kankrej calves were randomly divided into three treatment groups, viz. Basal diet (control), Basal diet+5 g animal-1 day-1 of fennel seed powder supplementation and Basal diet+10 g animal-1 day-1 of fennel seed powder supplementation for a period of 60 days. Results revealed that there was numerically higher body weight gains were observed in groups fed 5 and 10 g of fennel seed daily than the control, the difference was statistically not significant (p&gt;0.05) among the treatment groups. The mean faecal counts (log10 cfu g-1 in fresh faeces) of lactobacillus were increased, while coliform were reduced in groups fed 5 and 10 g of fennel seed daily as compared to control group. There was no effect (p&gt;0.05) on the haemoglobin, haematocrit, total erythrocytes count and total leucocytes count among the treatment groups. No difference (p&gt;0.05) was found in serum levels of total proteins, albumin, globulin, urea, triglycerides, cholesterol, ALT and AST concentrations among the treatment groups. It may be concluded that dietary fennel seed powder supplementation in calves improved growth performance and had beneficial effect on faecal microbiota with higher faecal lactobacillus count and lower faecal coliform count. Fennel seed supplementation did not affect haemato-biochemical profile of calves.

Pattern of Antibiotic Resistance in Salmonella sp. Bacteria Contaminating Fresh Faeces of Laying Hens in Kediri District, West Lombok Regency

Pattern of Antibiotic Resistance in Salmonella sp. Bacteria Contaminating Fresh Faeces of Laying Hens in Kediri District, West Lombok Regency

Phosphorus in farm roadway substrates: contrasting spatial and temporal patterns in dairy and beef farms

The mobilisation and impact of roadway runoff on dairy farms has been established as a sub-component of the nutrient transfer continuum. It is acknowledged that fresh nutrient sources deposited on roadways dominate runoff and recent work has shown that available phosphorus (P) in roadway substrates is also an important source component. The objectives of this study were to understand spatial and temporal available P concentrations in roadway substrate (aggregate and soil mix) across dairy and beef farms during the open (February–October, when cows use the roadway network and fields) and closed (November–January, when cows were housed) periods and identify locations which could be considered critical source areas (CSA). For the study, roadway substrate samples were taken at 18 locations on each farm, across eight sampling periods, and were analysed for available P. Results showed that mean available P concentrations (Morgan’s P) in the sampling locations ranged from 15.9 to 101.4 mg L-1 for the Dairy Farm, from 4.1 to 59.4 mg L-1 for Beef Farm 1 and from 6.3 to 23.2 mg L-1 for Beef Farm 2. In open period, the results showed that mean available P concentrations were 75.4 ± 30.9, 14.9 ± 3.2 and 13.4 ± 2.5 mg L-1 for the Dairy Farm, Beef Farm 1 and Beef Farm 2, respectively. In closed period, the mean available P were 40.3 ± 17.2, 10.4 ± 1.0 and 9.8 ± 0.2 mg L-1 for Dairy Farm, Beef Farm 1 and Beef Farm 2, respectively. Overall, P concentrations on the Dairy Farm roadways was up to 4 times greater than that in the Beef Farms’ roadways. Compared to soils in adjacent fields, P concentrations in the Dairy and Beef Farms roadways was up-to 7 and 2 times higher, respectively. Critical roadway sections that required mitigation were two in the Dairy Farm, one in Beef Farm 1 and two in Beef Farm 2. In addition to fresh faeces and urine (i.e., dominant source of nutrients in farm roadways), this study showed that nutrient enriched roadway materials are a labile P source. Thus, future mitigation of roadway runoff must consider all roadway nutrient sources, including livestock fresh excreta as well as used roadway surface materials.