Construction of recombinant fluorescent LSDV for high-throughput screening of antiviral drugs.

Lumpy skin disease virus (LSDV) infection is a major socio-economic issue that seriously threatens the global cattle-farming industry. Here, a recombinant virus LSDV-ΔTK/EGFP, expressing enhanced green fluorescent protein (EGFP), was constructed with a homologous recombination system and applied to the high-throughput screening of antiviral drugs. LSDV-ΔTK/EGFP replicates in various kidney cell lines, consistent with wild-type LSDV. The cytopathic effect, viral particle morphology, and growth performance of LSDV-ΔTK/EGFP are consistent with those of wild-type LSDV. High-throughput screening allowed to identify several molecules that inhibit LSDV-ΔTK/EGFP replication. The strong inhibitory effect of theaflavin on LSDV was identified when 100 antiviral drugs were screened in vitro. An infection time analysis showed that theaflavin plays a role in the entry of LSDV into cells and in subsequent viral replication stages. The development of this recombinant virus will contribute to the development of LSDV-directed antiviral drugs and the study of viral replication and mechanisms of action.

Surveillance of wild animals carrying infectious agents based on high-throughput screening platform in the Republic of Korea.

BACKGROUND: Infectious diseases transmitted by wild animals are major threats to public health. This study aimed to investigate the potential of rescued wild animals that died of unknown causes as reservoirs of infectious agents. From 2018 to 2019, 121 dead wild animals (55 birds and 66 mammals) were included in this study. All wild animals died during treatment after anthropogenic events. After deaths of animals, necropsies were performed and trachea, lungs, large intestine (including stool), and spleen were collected to determine causes of deaths. A high-throughput screening (HTS) quantitative polymerase chain reaction (qPCR) designed to detect 19 pathogens simultaneously against 48 samples in duplicate was performed using nucleic acids extracted from pooled tissues and peripheral blood samples. If positive, singleplex real-time PCR was performed for individual organs or blood samples. RESULTS: The HTS qPCR showed positive results for Campylobacter jejuni (10/121, 8.3%), Campylobacter coli (1/121, 0.8%), Mycoplasma spp. (78/121, 64.5%), and Plasmodium spp. (7/121, 5.7%). Singleplex real-time PCR confirmed that C. jejuni was detected in the large intestine but not in the blood. C. coli was only detected in the large intestine. Mycoplasma spp. were detected in all organs, having the highest proportion in the large intestine and lowest in the blood. Plasmodium spp. was also detected in all organs, with proportions being were similar among organs. CONCLUSIONS: This study shows that wild animals can become carriers of infectious agents without showing any clinical symptoms.

Histopathology is required to identify and characterize myopathies in high-throughput phenotype screening of genetically engineered mice.

The development of mouse models that replicate the genetic and pathological features of human disease is important in preclinical research because these types of models enable the completion of meaningful pharmacokinetic, safety, and efficacy studies. Numerous relevant mouse models of human disease have been discovered in high-throughput screening programs, but there are important specific phenotypes revealed by histopathology that are not reliably detected by any other physiological or behavioral screening tests. As part of comprehensive phenotypic analyses of over 4000 knockout (KO) mice, histopathology identified 12 lines of KO mice with lesions indicative of an autosomal recessive myopathy. This report includes a brief summary of histological and other findings in these 12 lines. Notably, the inverted screen test detected muscle weakness in only 4 of these 12 lines (Scyl1, Plpp7, Chkb, and Asnsd1), all 4 of which have been previously recognized and published. In contrast, 6 of 8 KO lines showing negative or inconclusive findings on the inverted screen test (Plppr2, Pnpla7, Tenm1, Srpk3, Sidt2, Yif1b, Mrs2, and Pnpla2) had not been previously identified as having myopathies. These findings support the need to include histopathology in phenotype screening protocols in order to identify novel genetic myopathies that are not clinically evident or not detected by the inverted screen test.

High-throughput viral microneutralization method for feline coronavirus using image cytometry.

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.

High-throughput sperm assay using label-free microscopy: morphometric comparison between different sperm structures of boar and stallion spermatozoa.

The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our knowledge, this is the first reported comparison when using AFM and high-sensitivity interferometric microscopy (such as SLIM) to evaluate spermatozoa. Results indicate that with the use of SLIM microscopy there is similar nanoscale sensitivity as with use of AFM while there is approximately 1,000 times greater throughput with use of SLIM. With SLIM, there is also allowace for the measurement of the dry mass (non-aqueous content) of spermatozoa, which may be a new label-free marker for sperm viability. In the second part of this study, there was analysis of two sperm populations. There were interesting correlations between the different compartments of the sperm and the dry mass in both boars and stallions. Furthermore, there was a correlation between the dry mass of the sperm head and the length and width of the acrosome in both boars and stallions. This correlation is positive in boars while it is negative in stallions.

Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing.

Tenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fish disease. This filamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft-sequenced genomes from isolates selected based on available MLST data, geographical origin and host fish. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein-coding genes accounting for ~75% of the genes in each genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp-1) whose analysis reveals an important contribution of recombination (r/m ≥ 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends between these subgroups and specific geographical origin or ecological niche remains to be clarified. We also evaluated the potential of MALDI-TOF-MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribosomal proteins explaining the detected mass shifts were identified. By combining nine polymorphic biomarker ions, we identified combinations referred to as MALDI-Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identified twenty MALDI-Types as well as four MALDI-Groups (MGs). We propose this MALDI-TOF-MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large-scale epidemiological surveys.

A high-throughput and broad-spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography-high-resolution mass spectrometry.

A high-throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide-conjugated drug targets. This is followed by extraction using solid-supported liquid extraction (SLE) and analysis using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The entire set-up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC-HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography-triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post-race or out-of-competition urine samples collected from racehorses. This method was developed for pre-race urine testing in Hong Kong; however, it is also suitable for testing post-race or out-of-competition urine samples, especially when a quick total analysis time is desired.

Combination of multiplex reverse transcription recombinase polymerase amplification assay and capillary electrophoresis provides high sensitive and high-throughput simultaneous detection of avian influenza virus subtypes.

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.

Detection of porcine epidemic diarrhea virus-neutralizing antibody using high-throughput imaging cytometry.

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry-based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status (n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

High-throughput quantification of protein structural change reveals potential mechanisms of temperature adaptation in Mytilus mussels.

BACKGROUND: Temperature exerts a strong influence on protein evolution: species living in thermally distinct environments often exhibit adaptive differences in protein structure and function. However, previous research on protein temperature adaptation has focused on small numbers of proteins and on proteins adapted to extreme temperatures. Consequently, less is known about the types and quantity of evolutionary change that occurs to proteins when organisms adapt to small shifts in environmental temperature. In this study, these uncertainties were addressed by developing software that enabled comparison of structural changes associated with temperature adaptation (hydrogen bonding, salt bridge formation, and amino acid use) among large numbers of proteins from warm- and cold-adapted species of marine mussels, Mytilus galloprovincialis and Mytilus trossulus, respectively. RESULTS: Small differences in habitat temperature that characterize the evolutionary history of Mytilus mussels were sufficient to cause protein structural changes consistent with temperature adaptation. Hydrogen bonds and salt bridges that increase stability and protect against heat-induced denaturation were more abundant in proteins from warm-adapted M. galloprovincialis compared with proteins from cold-adapted M. trossulus. These structural changes were related to deviations in the use of polar and charged amino acids that facilitate formation of hydrogen bonds and salt bridges within proteins, respectively. Enzymes, in particular those within antioxidant and cell death pathways, were over-represented among proteins with the most hydrogen bonds and salt bridges in warm-adapted M. galloprovincialis. Unlike extremophile proteins, temperature adaptation in Mytilus proteins did not involve substantial changes in the number of hydrophobic or large volume amino acids, nor in the content of glycine or proline. CONCLUSIONS: Small shifts in organism temperature tolerance, such as that needed to cope with climate warming, may result from structural and functional changes to a small percentage of the proteome. Proteins in which function is dependent on large conformational change, notably enzymes, may be particularly sensitive to temperature perturbation and represent foci for natural selection. Protein temperature adaptation can occur through different types and frequencies of structural change, and adaptive mechanisms used to cope with small shifts in habitat temperature appear different from mechanisms used to retain protein function at temperature extremes.

A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocapsid Np, glycoprotein Gn, and non-structural protein NSs. Sheep and cattle sera from a region in Kenya with previous outbreaks were tested by FMIA and two commercially available competitive ELISAs (BDSL and IDvet). Our results revealed strong detection of RVFV antibodies against the Np, Gn and NSs antigen targets. Additionally, testing of samples with FMIA Np and Gn had 100% agreement with the IDvet ELISA. The targets developed in the FMIA assay provided a basis for a larger ruminant disease panel that can simultaneously screen several abortive and zoonotic pathogens.

A high-throughput method for unbiased quantitation and categorization of nuclear morphology†.

The physical arrangement of chromatin in the nucleus is cell type and species-specific, a fact particularly evident in sperm, in which most of the cytoplasm has been lost. Analysis of the characteristic falciform ("hook shaped") sperm in mice is important in studies of sperm development, hybrid sterility, infertility, and toxicology. However, quantification of sperm shape differences typically relies on subjective manual assessment, rendering comparisons within and between samples difficult. We have developed an analysis program for morphometric analysis of asymmetric nuclei and characterized the sperm of mice from a range of inbred, outbred, and wild-derived mouse strains. We find that laboratory strains have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred strains, and that sperm shape in F1 offspring from a cross between CBA and C57Bl6J strains is subtly affected by the direction of the cross. We further show that hierarchical clustering can discriminate distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors, and is useful both to distinguish between samples and also to identify different morphological classes within a single sample. Our approach allows for the analysis of nuclear shape with unprecedented precision and scale and will be widely applicable to different species and different areas of biology.

High-throughput identification and quantification of Haemonchus contortus in fecal samples.

Haemonchus contortus are gastrointestinal nematodes of the family Trichostrongylidae that naturally infect small ruminants while grazing, posing a risk to both animal health and farm profitability. Current diagnostics depend on exacting lab techniques, including manual egg counts and larval differentiation, all of which require time, effort, and specialized technicians. The goal of this study was to facilitate and accelerate the identification and quantification of H. contortus in fecal samples through the use of fluorescein-isothiocyanate peanut-agglutinin staining in order to allow automated detection using a 96-well microplate reader. Next, the model was to be validated using samples containing unknown quantities of eggs. Automated analysis of fluorescence emission of known quantities of H. contortus eggs confirmed an almost perfect linear correlation (r = 0.9984, p < 0.0001), indicating that this new approach can satisfactorily be used to quantify H. contortus eggs on a comparative fluorescence scale. As validation, clinical samples containing an unknown quantity of H. contortus eggs were then analyzed by comparing two methods: either Wisconsin Sugar Flotation (WSF) and McMaster counting followed by manual fluorescence microscopy, or WSF coupled with automated microplate reading. Pearson analysis revealed highly significant correlation between manual and automated methods (r = 0.9999, p < 0.0001), while Bland-Altman plots demonstrated excellent agreement between the two (bias = -0.817 ± 9.94 with 95% limits of agreement from -20.31 to 18.67). Overall, these results demonstrate that high-throughput screening fluorescence detection and quantification of H. contortus eggs is both accurate and rapid.

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance.

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10-7. Based on the peak CT values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.

Involvement of selected cellular miRNAs in the in vitro and in vivo infection of infectious salmon anemia virus (ISAV).

Infectious salmon anemia virus (ISAV) is the causative agent of infectious salmon anemia (ISA), a relatively novel disease primarily affecting farmed salmon species, primarily in Salmo salar specimens, causing severe outbreaks in most producer countries. Although ISAV has been extensively studied at the molecular level, not much is known about the host/cell interaction at the small RNA level. MicroRNAs (miRNAs) are small, non-coding RNA that regulate mRNA expression at the post-transcriptional level. In recent years, the putative role of these molecules in host-pathogen interactions has drawn particular attention because of their pivotal involvement as regulatory elements in a number of eukaryotic organisms. Given the importance of the salmon industry in Chile, a deep understanding of the interaction between ISAV and its hosts is of importance. In the present work, we studied the kinetic expression of selected miRNAs during ISAV infection, both in vitro and in vivo. Based on initial experimental data derived from a small RNA-Seq analysis, a group of miRNAs that were differentially expressed in infected cells were selected for analysis. As a result, two miRNAs, miR-462a-5p and miR-125 b-5p, showed increased and decreased expression, respectively, during ISAV infection.

Global proteomic analysis of water buffalo (Bubalus bubalis) saliva at different stages of estrous cycle using high throughput mass spectrometry.

Accurate and efficient detection of estrus is one of the major constraints for exploitation of the production potential of buffalo owing to its poor manifestation of estrus signs, seasonal differences in expression and higher incidences of silent estrus (29%). The current study focused on identification of estrus specific candidate proteins in saliva of buffaloes. Estrus was detected based on behavioral signs in response to the teaser and changes in reproductive organs and confirmed by per-rectal examination, trans-rectal USG of reproductive organs, cervico-vaginal mucus characteristics and blood serum progesterone estimation. Day of onset of estrus was considered as day 0 and day -3, +3, +10 were considered as proestrus, metestrus and diestrus stage of the estrous cycle respectively. A total of 19 animals and their 38 estrous cycles (two from each) were included in this study. Saliva was collected from these animals during different stages of estrous cycle. Out of these, 08 animals were selected for global proteome analysis of saliva using in-solution digestion and nano-LC-MS/MS. A total of 275, 371, 304 and 565 proteins were identified with ≥2 peptides during proestrus, estrus, metestrus and diestrus stages of estrous cycle. Among the identified proteins 31, 62, 32 and 104 proteins were found specific to proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Few salivary proteins such as Cullin-associated NEDD8-dissociated protein 1, Heat shock 70 kDa protein 1A, 17-beta-hydroxysteroid dehydrogenase type 1, Inhibin beta A chain, testin were identified as estrus specific and are important for estrus physiology. Taken together, these estrus specific proteins could be considered as the candidate biomarker for detection and confirmation of estrus in buffalo after thorough validation.

In silico identification and high throughput screening of antigenic proteins as candidates for a Mannheimia haemolytica vaccine.

This study examined the use of comparative genomic analysis for vaccine design against Mannheimia haemolytica, a respiratory pathogen of ruminants. A total of 2,341genes were identified in at least half of the 23 genomes. Of these, a total of 240 were identified to code for N-terminal signal peptides with diverse sub-cellular localizations (78 periplasmic, 52 outer membrane, 15 extracellular, 13 cytoplasmic membrane and 82 unknown) and were examined in an ELISA assay using a coupled-cell free transcription/translation system for protein expressionwith antisera from cattle challenged with serovars 1, 2 or 6 of M. haemolytica. In total, 186 proteins were immunoreactive to at least one sera type and of these, 105 were immunoreactive to all sera screened. The top ten antigens based on immunoreactivity were serine protease Ssa-1 (AC570_10970), an ABC dipeptid transporter substrate-binding protein (AC570_04010), a ribonucleotide reductase (AC570_10780), competence protein ComE (AC570_11510), a filamentous hemagglutinin (AC570_01600), a molybdenum ABC transporter solute-binding protein (AC570_10275), a conserved hypothetical protein (AC570_07570), a porin protein (AC569_05045), an outer membrane assembly protein YeaT (AC570_03060), and an ABC transporter maltose binding protein MalE (AC570_00140). The framework generated from this research can be further applied towards rapid vaccine design against other pathogens involved in complex respiratory infections in cattle.

High-throughput sequencing technology to reveal the composition and function of cecal microbiota in Dagu chicken.

BACKGROUND: The chicken gut microbiota is an important and complicated ecosystem for the host. They play an important role in converting food into nutrient and energy. The coding capacity of microbiome vastly surpasses that of the host's genome, encoding biochemical pathways that the host has not developed. An optimal gut microbiota can increase agricultural productivity. This study aims to explore the composition and function of cecal microbiota in Dagu chicken under two feeding modes, free-range (outdoor, OD) and cage (indoor, ID) raising. RESULTS: Cecal samples were collected from 24 chickens across 4 groups (12-w OD, 12-w ID, 18-w OD, and 18-w ID). We performed high-throughput sequencing of the 16S rRNA genes V4 hypervariable regions to characterize the cecal microbiota of Dagu chicken and compare the difference of cecal microbiota between free-range and cage raising chickens. It was found that 34 special operational taxonomic units (OTUs) in OD groups and 4 special OTUs in ID groups. 24 phyla were shared by the 24 samples. Bacteroidetes was the most abundant phylum with the largest proportion, followed by Firmicutes and Proteobacteria. The OD groups showed a higher proportion of Bacteroidetes (>50 %) in cecum, but a lower Firmicutes/Bacteroidetes ratio in both 12-w old (0.42, 0.62) and 18-w old groups (0.37, 0.49) compared with the ID groups. Cecal microbiota in the OD groups have higher abundance of functions involved in amino acids and glycan metabolic pathway. CONCLUSION: The composition and function of cecal microbiota in Dagu chicken under two feeding modes, free-range and cage raising are different. The cage raising mode showed a lower proportion of Bacteroidetes in cecum, but a higher Firmicutes/Bacteroidetes ratio compared with free-range mode. Cecal microbiota in free-range mode have higher abundance of functions involved in amino acids and glycan metabolic pathway.

Evaluation of high-throughput assays for in vitro drug susceptibility testing of Tritrichomonas foetus trophozoites.

Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes abortions in cattle and results in severe economic losses. In the United States, there are no safe and effective treatments for this parasite and infected animals are typically culled. In order to expedite drug discovery efforts, we investigated in vitro trophozoite killing assays amenable to high-throughput screening in 96 well plate formats. We evaluated the reduction of resorufin, incorporation of propidium iodide, and a luminescence-based ATP detection assay. Of these methods, reduction of resorufin was found to be the most reliable predictor of trophozoite concentrations. We further validated this method by conducting dose-response experiments suitable for calculation of EC50 values for two established compounds with known activity against trophozoites in vitro, namely, metronidazole and ronidazole. Our results demonstrate that the resorufin method is suitable for high-throughput screening and could be used to enhance efforts targeting new treatments for bovine trichomoniasis.