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Background: Congenital transmission (CT) of Trypanosoma cruzi in dogs has not been clearly demonstrated, even though dogs are important reservoirs of this agent. Materials and Methods: Seventeen late pregnant dogs seropositive for T. cruzi were selected, and a total of 84 fetuses were obtained. Blood and heart tissues from the fetuses and dams, and placental tissue from dam were collected. All tissues were analyzed by quantitative polymerase chain reaction (qPCR) for T. cruzi DNA (TcDNA) and inflammatory infiltrate and pathology by histological examination. CT was determined when physical, histological, or molecular evidence of T. cruzi was detected in blood or tissues of the fetuses. Results: A general transmission frequency of 59% was found, and 0.20 ± 0.24 of fetuses per litter were infected. Dams that were qPCR positive for TcDNA in cardiac tissue or blood displayed a transmission frequency of 100% and 67%, respectively. The highest parasite burden was noted in dams that were positive for TcDNA in both blood (82E-01 ± 1.54E-01) and cardiac (5.28E+03 ± 8.85E+03) tissues. In fetuses, higher parasitic burden in blood and cardiac tissue was found in those carried by dams that were seropositive and qPCR positive for TcDNA in cardiac tissue and blood. No amastigote nests were recorded in the cardiac tissue of fetuses in the histopathological studies, but typical lesions of T. cruzi infection were identified in all fetuses where CT occurred. Conclusions: CT of T. cruzi occurred at a high frequency in naturally infected pregnant dogs from the endemic areas. [ABSTRACT FROM AUTHOR]

The fowl glioma-inducing virus prototype (FGVp) and its variants, which belong to avian leukosis virus subgroup A (ALV-A), induce cardiomyocyte abnormalities and gliomas in chickens. However, the molecular mechanisms underlying these myocardial changes remain unclear, and ALV-induced tumorigenesis, which is caused by proviral insertional mutagenesis, does not explain the early development of cardiac changes in infected chickens. We established a quantitative PCR (qPCR) assay to measure ALV-A proviral loads in the brains and hearts of FGV-infected Japanese bantam chickens and compared these results with morphologic lesions. Four of 22 bantams had both gliomas and cardiac lesions. Hearts with cardiac lesions had a higher proviral load (10.3 ± 2.7 proviral copies/nucleus) than those without cardiac lesions (0.4 ± 0.4), suggesting that the proviral load in hearts is correlated with the frequency of myocardial changes. Our qPCR method may be useful in the study of ALV-induced cardiomyocyte abnormalities. [ABSTRACT FROM AUTHOR]

The microbiota in gastrointestinal tracts is recognized to play a pivotal role in the health of their hosts. Music and noise are prevalent environmental factors in human society and animal production and are reported to impact their welfare and physiological conditions; however, the information on the relationship between the microbiota, physiological status, and sound is limited. This study investigated the impact of music and white noise exposure in mice through 16s rRNA gene sequencing, enzyme assay, and qPCR. The results demonstrate that white noise induced oxidative stress in animals by decreasing serum SOD and GSH-PX activity while increasing LDH activity and MDA levels (p < 0.05). Conversely, no oxidative stress was observed in the music treatment group. The relative gene expression of IFN-γ and IL-1β decreased in the white noise group compared to the music and control groups. The 16s rRNA gene amplicon sequencing revealed that Bacteroidetes, Firmicutes, Verrucomicrobia, and Proteobacteria were dominant among all the groups. Furthermore, the proportion of Firmicutes increased in the music treatment group but decreased in the white noise treatment group compared to the control group. In conclusion, white noise has detrimental impacts on the gut microbiota, antioxidant activity, and immunity of mice, while music is potentially beneficial. [ABSTRACT FROM AUTHOR]

Panulirus argus virus 1 (PaV1) is the first and only naturally occurring pathogenic virus described in the Caribbean spiny lobster, Panulirus argus. PaV1 infection in decapod species that commonly co-occur with P. argus, including the spotted spiny lobster Panulirus guttatus, has not been previously described. In 2016, 14 Caribbean and 5 spotted spiny lobsters were collected near Summerland Key, Florida, to supplement the resident population of the Audubon Aquarium of the Americas in New Orleans, Louisiana. After 5 months in quarantine, Caribbean and spotted spiny lobsters began to exhibit clinical signs of lethargy and dying in the molt. Initial histologic evaluation revealed intranuclear inclusion bodies in circulating hemocytes in the spongy connective tissue of the epidermis, suggesting a viral infection. Samples of hepatopancreas and hemolymph from deceased Caribbean and spotted spiny lobsters tested negative for white spot syndrome virus and positive for PaV1 using real-time quantitative polymerase chain reaction (qPCR). Intranuclear, eosinophilic to amphophilic, Cowdry type A inclusion bodies observed primarily within fixed phagocytes and circulating hemocytes in the hepatopancreas of freshly euthanized Caribbean spiny lobsters were consistent with PaV1 infection. Transmission electron microscopy revealed that hemocytes associated with hepatopancreatic tubules contained viral inclusions with location, size, and morphology consistent with previously described PaV1 infection. These findings highlight the significance of using molecular diagnostics in conjunction with histopathology and electron microscopy in the investigation and diagnosis of PaV1 in spiny lobsters. Further study is required to investigate the relationship of PaV1-associated mortality events and microscopic lesions in the spotted spiny lobster. [ABSTRACT FROM AUTHOR]

Simple Summary: African swine fever virus is a pathogen capable of spreading among swine populations, which is usually fatal since no vaccines are currently available on the market. Outbreaks caused by this virus are the reason for massive economic losses on pig farms and are a matter of worry for the swine sector worldwide. Effective and reliable detection of the virus is relevant to prevent uncontrolled contagion, especially when no effective preventive measures can be applied. In order to confront the spread of African swine fever, several rapid real-time polymerase chain reaction detection assays were developed by different research groups. In the present study, we focused on the validation of the ASFV MONODOSE dtec-qPCR kit using reference genetic material provided by national and international reference laboratories, and results were compared with those obtained from using internationally accepted reference methods. The data obtained indicated that the kit can be used in veterinary samples. Technical innovations enable a user-friendly and rapid management of samples, reducing the probability of human errors happening and transport costs. African swine fever virus is considered an emerging virus that causes African swine fever, a disease characterised by high mortality and elevated transmission rates and that, as it is for most other viral diseases, cannot be treated with specific drugs. Effective and reliable detection of the virus is relevant to prevent uncontrolled contagion among boar populations and to reduce economic losses. Moreover, animal health laboratories are demanding standardisation, optimisation and quality assurance of the available diagnostic assays. In the present study, the ASFV MONODOSE dtec-qPCR kit was validated following the UNE-EN ISO/IEC 17025:2005 guidelines. Analytical validation terms include in silico and in vitro specificity, sensitivity, efficiency and reliability (repeatability/reproducibility). Diagnostic validation of the method was assessed through the analysis of a total of 181 porcine samples originating from six different matrix types doped with African swine fever virus DNA received from the European reference laboratory for African Swine Fever (INIA-CISA, Madrid, Spain): whole blood, blood serum, kidney, heart, liver and tonsil. Results agreed with those obtained from a reference detection method also based on real-time PCR, endorsed by WOAH, but the ASFV MONODOSE dtec-qPCR kit incorporates some technical innovations and improvements which may benefit end-users. This kit, available worldwide with full analytical and diagnostic validation, can recognise all known ASFV genotypes and brings additional benefits to the current qPCR technology. [ABSTRACT FROM AUTHOR]

PI-PLC is not only involved in the regulation of plant responses to a variety of biotic and abiotic stresses, but also plays an important role in the regulation of plant growth. Banana is one of the four most famous fruits in the world, as well as one of the most important tropical fruits in the world. However, in recent years, blight and cold damage have become two major deadly hazards for banana industry, making it particularly important to improve the resistance of bananas to stress. In this study, PI-PLCs gene family of banana encodes 567–698 amino acids. It is mainly located in chloroplasts, mitochondria and cytoplasm. Systematic evolution and homology analysis, PI-PLCs gene family of banana is highly clustered, the structure and function are relatively consistent. There were hormones and stress response elements such as methyl jasmonate, auxin and gibberellin, in the promoter region of MaPLCs family members. The proteins coded by all MaPLCs could interact and participate in a series of life-sustaining activities. The study showed that members of the MaPLCs gene family of bananas could be induced to express by auxin, abscisic acid and salicylic acid. [ABSTRACT FROM AUTHOR]

Fulminant hepatitis is a severe clinical disease characterized by a marked decline in liver function and encephalopathy. In a previous survey, using metagenomics in a group of 27 patients with this clinical condition, we observed an expressive quantity of reads of the Human pegivirus-1 (HPgV-1). Therefore, the objective of this study was to evaluate the frequency, molecular features, and HPgV-1 circulating genotypes in patients with fulminant hepatitis. After testing the collected plasma samples, we discovered twelve samples (44.4%) that were positive for HPgV-1 RNA (using both real-time and nested PCR). The positive samples presented a mean cycle threshold (Ct) of 28.5 (±7.3). Genotyping assignments revealed that all HPgV-1 positive samples belonged to the HPgV-1 genotype 2 (both subgenotypes 2A and 2B were identified). Although HPgV-1 is considered a commensal virus, little is known regarding its prevalence and genotypes in cases of fulminant hepatitis. More research is needed to understand whether HPgV-1 can be implicated in clinical disorders and infectious diseases. [ABSTRACT FROM AUTHOR]

Background and aims: Solid tumors account for about 30% of all pediatric cancers. The diagnosis is typically based on histological and molecular analysis of a primary tumor biopsy. Liquid biopsies carry several advantages over conventional tissue biopsy. However, their use for genomic analysis and response monitoring of pediatric solid tumors is still in experimental stages and mostly performed retrospectively without direct impact on patient management. In this case series we discuss six clinical cases of children with a solid tumor for whom a liquid biopsy assay was performed and demonstrate the potential of liquid biopsy for future clinical decision making. Methods: We performed quantitative real-time PCR (RT-qPCR), droplet digital PCR (ddPCR) or reduced representation bisulphite sequencing of cell-free DNA (cfRRBS) on liquid biopsies collected from six pediatric patients with a solid tumor treated between 2017 and 2023 at the Princess Maá xima Center for Pediatric Oncology in the Netherlands. Results were used to aid in clinical decision making by contribution to establish a diagnosis, by prognostication and response to therapy monitoring. Results: In three patients cfRRBS helped to establish the diagnosis of a rhabdomyosarcoma, an Ewing sarcoma and a neuroblastoma (case 1-3). In two patients, liquid biopsies were used for prognostication, by MYCN ddPCR in a patient with neuroblastoma and by RT-qPCR testing rhabdomyosarcomaspecific mRNA in bone marrow of a patient with a rhabdomyosarcoma (case 4 and 5). In case 6, mRNA testing demonstrated disease progression and assisted clinical decision making. Conclusion: This case series illustrates the value of liquid biopsy. We further demonstrate and recommend the use of liquid biopsies to be used in conjunction with conventional methods for the determination of metastatic status, prognostication and monitoring of treatment response in patients with pediatric solid tumors. [ABSTRACT FROM AUTHOR]

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The dissemination of antibiotic resistance genes (ARGs) in the environment contributes to the global rise in antibiotic resistant infections. Therefore, it is of importance to further research the exposure pathways of these emerging contaminants to humans. This study explores commercially available garden products containing animal manure as a source of ARGs in a survey of 34 garden products, 3 recently landscaped soils, and 5 native soils. DNA was extracted from these soils and quantified for 5 ARGs, intI1, and 16S rRNA. This study found that both absolute and relative gene abundances in garden products ranged from approximately two to greater than four orders of magnitude higher than those observed in native soils. Garden products with Organic Materials Review Institute (OMRI) certification did not have significantly different ARG abundances. Results here indicate that garden products are important sources of ARGs to gardens, lawns, and parks.

This study investigated the effect of krill oil (KO) on liver damage caused by acetaminophen (APAP).

Abstract Environmental DNA (eDNA) once shed can exist in numerous states with varying behaviors including degradation rates and transport potential. In this study, we consider three states of eDNA: (1) a membrane‐bound state referring to DNA enveloped in a cellular or organellar membrane, (2) a dissolved state defined as the extracellular DNA molecule in the environment without any interaction with other particles, and (3) an adsorbed state defined as extracellular DNA adsorbed to a particle surface in the environment. Capturing, isolating, and analyzing a target state of eDNA provides utility for better interpretation of eDNA degradation rates and transport potential. While methods for separating different states of DNA have been developed, they remain poorly evaluated due to the lack of state‐controlled experimentation. We evaluated the methods for separating states of eDNA from a single sample by spiking DNA from three different species to represent the three states of eDNA as state‐specific controls. We used chicken DNA to represent the dissolved state, cultured mouse cells for the membrane‐bound state, and salmon DNA adsorbed to clay particles as the adsorbed state. We performed the separation in three water matrices, two environmental and one synthetic, spiked with the three eDNA states. The membrane‐bound state was the only state that was isolated with minimal contamination from nontarget states. The membrane‐bound state also had the highest recovery (54.11 ± 19.24%), followed by the adsorbed state (5.08 ± 2.28%), and the dissolved state had the lowest total recovery (2.21 ± 2.36%). This study highlights the potential to sort the states of eDNA from a single sample and independently analyze them for more informed biodiversity assessments. However, further method development is needed to improve recovery and reduce cross‐contamination.

Abstract Background Circular RNAs (circRNAs) are covalently closed-loop RNAs with critical regulatory roles in cells. Tens of thousands of circRNAs have been unveiled due to the recent advances in high throughput RNA sequencing technologies and bioinformatic tools development. At the same time, polymerase chain reaction (PCR) cross-validation for circRNAs predicted by bioinformatic tools remains an essential part of any circRNA study before publication. Results Here, we present the CircPrime web-based platform, providing a user-friendly solution for DNA primer design and thermocycling conditions for circRNA identification with routine PCR methods. Conclusions User-friendly CircPrime web platform ( http://circprime.elgene.net/ ) works with outputs of the most popular bioinformatic predictors of circRNAs to design specific circular RNA primers. CircPrime works with circRNA coordinates and any reference genome from the National Center for Biotechnology Information database).

【Objective】Hybrid snakehead is an important special aquatic fish in China. In order to find out the infection status of hybrid snakehead rhabdovirus (HSHRV), the rhabdovirus disease of hybrid snakehead cultured in the Pearl River Delta was investigated from March 2021 to February 2022.【Method】The parents, fry and water samples of hybrid snakehead were randomly sampled in breeding stage, and these were randomly sampled at fixed time in culture stage and sampled when the outbreak of the virus. A total of 333 samples were collected during the whole monitoring period. Mixed samples of mung bean-sized liver, spleen and kidney were stored in liquid nitrogen for RNA extraction. And reverse-transcribed cDNA was used as template to detect rhabdovirus by real-time fluorescence quantitative PCR (qPCR).【Result】The main clinical symptoms of rhabdovirus-infected fish were that the fish swam irregularly and spin. Although there was no obvious symptom on the body surface, the liver, spleen, kidney, intestine and swim bladder became red or swollen. The qPCR results revealed that the average positive rate of HSHRV was 13.21%, which was mainly detected from April to November. Among them, the positive rate of samples detected in August was as high as 23.91%. The average positive rate of HSHRV in the culture stage (17.01%) was significantly higher than that in the breeding stage (3.26%). In terms of fish body size, 32.20% of the fry were most susceptible to HSHRV infection within 10 cm, mainly detected from March to September, while 9.52% of the fish of more than 20 cm were least susceptible to HSHRV infection.【Conclusion】The positive rates of HSHRV in hybrid snakehead in breeding stage and cultivating stage as well as in different size are determined, which provide references for further understanding the epidemic regularity of hybrid snakehead rhabdovirus disease, with a view to providing data support for the prevention and control of hybrid snakehead rhabdovirus disease.

Abstract Background Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34. Results NSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNFα levels was seen in the infected neurons, which is of interest because TNFα protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron. Conclusions The findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.

Introduction: Heat and its effects are considered one of the serious threats and damages to the production of agricultural and horticultural crops in many regions of the world. Therefore, it has led to pervasive changes in terms of morphological, physiological, anatomical, and molecular, which causes a sharp decrease in the economic performance of agricultural products. Nowadays, in order to reduce these inhibitory effects, in addition to classical and conventional breeding methods, modern and molecular methods can be used to identify heat-tolerant genes and use them in breeding programs to improve plant tolerance to environmental stresses. Materials and methods: The selection for heat tolerance among four local cultivars Hovyzeh, Hamar, Red-Anbori, Champa and three promising heat tolerant lines V1, V2 and V3 obtained from the international breeding programs of Khuzestan Agricultural Research and Training Center and Natural Resources were evaluated by osHATS and HSP70 genes qPCR on two experimental cultivation dates: May 20 (unfavorable) and June 20 (favorable) 2022 in Ahvaz. Results: The results obtained from the analysis of gene expression by qPCR on the date of the first cultivation confirmed the presence of the osHTAS gene in rice varieties and lines so that the local variety Champa, Hovyzeh, and line V2 has a high relative gene expression compared to other varieties and lines studied and they were significantly different from each other. The analysis of the osHTAS gene expression on the second planting date (June) in Ahvaz also shows the presence of the high-temperature tolerance gene in Hovyzeh, Hamar and V2 lines; therefore, the mentioned varieties could be used as parental lines for transfer heat tolerance gene to be used in multi-breeding programs and terms of the relative expression level of the gene, they showed a statistically significant difference with each other. The results of the relative gene expression of the HSP70 gene in the cultivation date of May showed that the local cultivars Champa, Red-Anbori and Hovyzeh had a remarkable superiority over the others. Examining the HSP70 gene expression pattern of rice cultivars and lines on the second planting date also revealed that the local cultivars Red-Anbori, Champa, Hovyzeh, and Hamar showed superiority. The tolerance of local cultivars to heat stress is due to their rapid growth and the creation of a large green cover in the early stages of growth, and their great adaptation to the region's environmental conditions. Conclusion: Local cultivars can withstand heat stress well with rapid growth and creating much vegetation due to their high adaptability to environmental conditions and the presence of heat stress tolerance genes. They could be used as parental lines in breeding programs.

Abstract Background One of the major roadblocks to the falciparum malaria elimination programme is the presence of a portion of the population, such as school children, with asymptomatic malaria infection. Targeting such reservoirs of infections is critical to interrupting transmission and enhancing elimination efforts. The NxTek™ Eliminate Malaria Pf test is a highly sensitive rapid diagnostic test (hsRDT) for the detection of HRP-2. However, knowledge gaps exist in Ethiopia on the diagnostic performance of hsRDT for the detection of Plasmodium falciparum in school children with asymptomatic malaria. Methods A school-based cross-sectional study was conducted from September 2021 to January 2022 on 994 healthy school children (aged 6–15 years). Finger-pricked whole blood samples were collected for microscopy, hsRDT, conventional RDT (cRDT or SD Bioline Malaria Ag Pf/P.v), and QuantStudio™ 3 Real—Time PCR system (qPCR). The hsRDT was compared to cRDT and microscopy. qPCR and microscopy were used as reference methods. Results The prevalence of Plasmodium falciparum was 1.51%, 2.2%. 2.2% and 4.52%, by microscopy, hsRDT, cRDT and qPCR, respectively. Using qPCR as reference, the sensitivity of hsRDT was higher (48.89%) than the microscopy (33.3%), and showed 100% specificity and a positive predictive value (PPV). Microscopy showed similar specificity and PPV as hsRDT. Using microscopy as a reference, the diagnostic perforrmances of both hsRDT and cRDT were similar. Both RDTs demonstrated identical diagnostic performances in both comparison methods. Conclusions hsRDT has the same diagnostic performance as cRDT but improved diagnostic characteristics than microscopy for detection of P. falciparum in school children with asymptomatic malaria. It can be a useful tool for the national malaria elimination plan of Ethiopia.

Pneumonia is among the most serious manifestations of HCMV infection, with high morbidity and mortality. Probable pneumonia is defined as the detection of HCMV in bronchoalveolar lavage (BAL) by viral isolation or DNA quantification (qPCR) combined with symptoms and/or signs of respiratory infection. However, currently, there is no reproducible and well-defined viral load (VL) from BAL that can reliably differentiate patients with pneumonia from the much more common detection of viral DNA in seropositive patients without true HCMV pneumonia. Several studies have been published with the aim of establishing an optimal VL for differentiating pneumonia from viral lung shedding. The aim of this review is to collect and analyze the methodology and the conclusions obtained in studies whose objectives included the correlation between HCMV VL in BAL and/or the plasma and the occurrence of HCMV pneumonia. For this purpose, a total of 14 articles have been included. There are some conclusions on which they all agree. PCR techniques were more sensitive and had a higher NPV than culture techniques but were less specific and had a low PPV. The mean HCMV loads in both BAL and the plasma were significantly higher in patients with pneumonitis than in those without. The HCMV load in patients with pneumonitis was higher in BAL than in the plasma, making qPCR in BAL a better predictor of HCMV pneumonitis than in the plasma. Nevertheless, this review highlights the difficulty of establishing a universal VL value, both in BAL and in the blood, to differentiate patients with HCMV pneumonia from those without. To complete the information available in these studies, prospective multicentre studies would be required. Methodologically, a large number of patients with HCMV pneumonitis would have to be included, and a subclassification of the type of immunosuppression of each patient should be made in order to obtain an optimal VL threshold in different host groups.

Here, a method using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER) for miRNAs quantification was established. The strategy has two steps: (1) ligation of two DNA probes specifically hybridize to target miRNA and (2) qPCR amplifying the ligated probe. The miRNA-binding regions of the probes are stem-looped, a motif significantly reduces nonspecific ligation at high ligation temperature (65°C). The ends of the probes are designed complementary to form a paired probe, facilitating the recognition of target miRNAs with low concentrations. RNase H proved to be able to stabilize the heteroduplex formed by the probe and target miRNA, contributing to enhanced sensitivity (limit of detection = 60 copies). High specificity (discriminating homology miRNAs differing only one nucleotide), wide dynamic range (seven orders of magnitude) and ability to accurately detect plant miRNAs (immune to hindrance of 2′-O-methyl moiety) enable SPLICER comparable with the commercially available TaqMan and miRCURY assays. SYBR green I, rather than expensive hydrolysis or locked nucleic acid probes indispensable to TaqMan and miRCURY assays, is adequate for SPLICER. The method was efficient (

A workflow is described for assaying the expression of G protein-coupled receptors (GPCRs) in cultured cells, using a combination of methods that assess GPCR mRNAs. Beginning from the isolation of cDNA and preparation of mRNA, we provide protocols for designing and testing qPCR primers, assaying mRNA expression using qPCR and high-throughput analysis of GPCR mRNA expression via TaqMan qPCR-based, GPCR-selective arrays. We also provide a workflow for analysis of expression from RNA-sequencing (RNA-seq) assays, which can be queried to yield expression of GPCRs and related genes in samples of interest, as well as to test changes in expression between groups, such as in cells treated with drugs or from healthy and diseased subjects. We place priority on optimized protocols that distinguish signal from noise, as GPCR mRNAs are typically present in low abundance, necessitating techniques that maximize sensitivity while minimizing noise. These methods may also be applicable for assessing the expression of members of families of other low abundance genes via high-throughput analyses of mRNAs, followed by independent confirmation and validation of results via qPCR.

Three newly identified equine parvoviruses (equine parvovirus hepatitis (EqPV-H), equine parvovirus CSF (EqPV-CSF) and equine copivirus (Eqcopivirus)) have recently been discovered in horses with respiratory signs. However, the clinical impact of these three equine parvoviruses has yet to be determined. Nasal fluid samples and blood from 667 equids with acute onset of fever and respiratory signs submitted to a diagnostic laboratory were analyzed for the presence of common equine respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, S. equi subspecies equi) as well as EqPV-H, EqPV-CSF and Eqcopivirus by qPCR. An additional 87 clinically healthy horses served as controls. One hundred and seventeen sick horses tested qPCR-positive for at least one of the three parvoviruses. Co-infections with common respiratory pathogens and parvoviruses were seen in 39 sick equids. All 87 clinically healthy horses tested qPCR-negative for all tested common respiratory pathogens and 10 healthy horses tested qPCR-positive for one of the equine parvoviruses. When the frequency of detection for EqPV-H, EqPV-CSF and Eqcopivirus of equids with respiratory signs was compared to that of clinically healthy horses, the difference was not statistically significant (p > 0.05), suggesting that the three recently identified equine parvoviruses do not contribute to the clinical picture of equids with respiratory disease.

The objectives of this study were to evaluate the prevalence of extended spectrum β-lactamase (ESBL) genes, AmpC-type β-lactamase (ACBL) genes, and plasmid mediated quinolone resistance (PMQR) genes in Salmonella isolated at a Veterinary Medical Teaching Hospital microbiology laboratory, examine trends in presence of these resistance genes, and to explore the correlation between phenotypic resistance and presence of specific genes. The presence of ESBL, ACBL, and PMQR genes were detected using a single, novel multiplex qPCR. Only the genes bla CMY-2 and bla TEM were detected in the 110 Salmonella isolates tested. PMQR genes were not detected in isolates screened. Of 94 third-generation cephalosporin resistant isolates, representing eight serotypes, 48% (n = 45) were positive for bla CMY-2 only and 50% (n = 47) were simultaneously positive for bla CMY-2 and bla TEM. Two third-generation cephalosporin resistant isolates were tested negative for all β-lactamase genes in our qPCR assay and likely house ESBL genes not screened for by our qPCR assay. A logistic regression model revealed that for serotype Dublin isolates (n = 38) the odds ratio for testing positive for bla TEM when compared to all other serotypes was 51.6 (95% CI: 4.01-664.03, p = 0.0029). For serotype Typhimurium (n = 9) the odds ratio for testing positive for bla TEM when compared to all other serotypes was 43.3 (95% CI: 1.76-1000, p = 0.0216). Overall, our results suggest that the prevalence of resistance to cephalosporins and fluoroquinolones due to ESBLs, ACBLs, and PMQR genes present in bovine nontyphoidal Salmonella enterica isolates has remained relatively constant in the isolates screened over a 14-year period.

Background and objectivesA previous pre-clinical study on electromagnetic muscle stimulation (EMMS) suggested that fat cell apoptosis occurs following treatment in a porcine model. While EMMS can induce changes in muscle, the effect on fat tissue is not established. This clinical study sought to assess adipose tissue response to EMMS in comparison to cryolipolysis treatment.Study design/materials and methodsStudy subjects were recruited prior to abdominoplasty to receive body contouring treatments and subsequently to obtain tissue for histological analysis. Non-invasive abdominal treatments were delivered using a commercially available (n = 6) or prototype (n = 3) EMMS system or a cryolipolysis system (n = 2). Subjects received a single EMMS treatment (100% intensity for 30 minutes) or a single cryolipolysis treatment (-11°C for 35 minutes) to the abdomen. Superficial and deep (i.e., adjacent to muscle layer) subcutaneous adipose tissue was harvested at set timepoints post-treatment. The presence or absence of an inflammatory response was evaluated using standard hematoxylin and eosin (H&E) staining. As adipocytes that are destined to become apoptotic cannot be distinguished by traditional H&E staining during the early phases of injury, irreversible fat cell injury was assessed using perilipin immunofluorescence.ResultsFollowing H&E histological analysis at 3, 10, 11, and 17 days post-treatment, no EMMS-treated samples showed an inflammatory response in either the superficial or deep subcutaneous adipose tissue. For the cryolipolysis-treated adipose tissue, however, the H&E staining revealed a marked inflammatory response with an influx of neutrophils, lymphocytes, and macrophages at timepoints consistent with previous histological studies. Further, loss of perilipin staining provided clear visual evidence of irreversible fat cell injury in the cryolipolysis-treated adipose tissue. In contrast, the electromagnetic muscle stimulation-treated samples showed persistence of perilipin staining of adipose tissue indicating that all fat cells were viable.ConclusionThis study failed to demonstrate either fat cell injury or inflammatory response following EMMS treatment. While electromagnetic muscle stimulation may non-invasively induce muscle changes, this clinical study found no evidence of an impact injurious or otherwise on subcutaneous fat. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8-100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9-99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5-100]; n = 37) and Se was 93.3% (95% CI [68.1-99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

Due to the COVID-19 pandemic and potential public health implications, we are publishing this peer-reviewed manuscript in its accepted form. The final, copyedited version of the paper will be available at a later date. Although SARS-CoV-2 is primarily transmitted by respiratory droplets and aerosols, transmission by fomites remains plausible. During Halloween, a major event for children in numerous countries, SARS-CoV-2 transmission risk via candy fomites worries many parents. To address this concern, we enrolled 10 recently diagnosed asymptomatic or mildly/moderately symptomatic COVID-19 patients to handle typical Halloween candy (pieces individually wrapped) under three conditions: normal handling with unwashed hands, deliberate coughing and extensive touching, and normal handling following handwashing. We then used a factorial design to subject the candies to two post-handling treatments: no washing (untreated) and household dishwashing detergent. We measured SARS-CoV-2 load by RT-qPCR and LAMP. From the candies not washed post-handling, we detected SARS-CoV-2 on 60% of candies that were deliberately coughed on, 60% of candies normally handled with unwashed hands, but only 10% of candies handled after hand washing. We found that treating candy with dishwashing detergent reduced SARS-CoV-2 load by 62.1% in comparison to untreated candy. Taken together, these results suggest that although the risk of transmission of SARS-CoV-2 by fomites is low even from known COVID-19 patients, viral RNA load can be reduced to near zero by the combination of handwashing by the infected patient and ≥1 minute detergent treatment after collection. We also found that the inexpensive and fast LAMP protocol was more than 80% concordant with RT-qPCR.IMPORTANCE The COVID-19 pandemic is leading to important tradeoffs between risk of SARS-CoV-2 transmission and mental health due to deprivation from normal activities, with these impacts being especially profound in children. Due to the ongoing pandemic, Halloween activities will be curtailed as a result of the concern that candy from strangers might act as fomites. Here we demonstrate that these risks can be mitigated by ensuring that prior to handling candy, the candy giver washes their hands, and by washing collected candy with household dishwashing detergent. Even in the most extreme case, with candy deliberately coughed on by known COVID-19 patients, viral load was reduced dramatically after washing with household detergent. We conclude that with reasonable precautions, even if followed only by either the candy giver or the candy recipient, the risk of viral transmission by this route is very low.

BackgroundNext generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.ResultsIn this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data.ConclusionsWe successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells.

Obstructive sleep apnea (OSA) is quite prevalent during pregnancy and is associated with adverse perinatal outcomes, but its potential influence on fetal development remains unclear. This study investigated maternal OSA impact on the fetus by analyzing gene expression profiles in whole cord blood (WCB). Ten women in the third trimester of pregnancy were included, five OSA and five non-OSA cases. WCB RNA expression was analyzed by microarray technology to identify differentially expressed genes (DEGs) under OSA conditions. After data normalization, 3238 genes showed significant differential expression under OSA conditions, with 2690 upregulated genes and 548 downregulated genes. Functional enrichment was conducted using gene set enrichment analysis (GSEA) applied to Gene Ontology annotations. Key biological processes involved in OSA were identified, including response to oxidative stress and hypoxia, apoptosis, insulin response and secretion, and placental development. Moreover, DEGs were confirmed through qPCR analyses in additional WCB samples (7 with OSA and 13 without OSA). This highlighted differential expression of several genes in OSA (EGR1, PFN1 and PRKAR1A), with distinct gene expression profiles observed during rapid eye movement (REM)-OSA in pregnancy (PFN1, UBA52, EGR1, STX4, MYC, JUNB, and MAPKAP). These findings suggest that OSA, particularly during REM sleep, may negatively impact various biological processes during fetal development.

Ca and P homeostasis across the egg-laying cycle is a complex process involving absorption in the small intestine, reabsorption/excretion in the kidneys, and eggshell gland secretion. Diets with inadequate calcium and phosphorus can interfere with their absorption and digestibility, resulting in eggshell quality losses and reduced productive life, affecting egg production and welfare. A better understanding of gene expression profiles in the kidneys of laying hens during the late egg-laying period could clarify the renal role in mineral metabolism at this late stage. Therefore, the performance, egg quality and bone integrity-related traits, and expression profiles of kidney candidate genes were evaluated in 73-week-old laying hens receiving different Ca and P ratios in their diet: a high Ca/P ratio (HR, 22.43), a low ratio (LR, 6.71), and a medium ratio (MR, 11.43). The laying hens receiving the HR diet had improved egg production and eggshell quality traits compared to the other two groups. Humerus length was shorter in the HR than in the other groups. The CYP24A1 and TRPC3 genes were differentially expressed (p.adj ≤ 0.05) among the groups. Therefore, their expression profiles could be involved in calcium and phosphate transcellular transport in 73-week-old laying hens as a way to keep mineral absorption at adequate levels.

DNA polymerase β (DNA polymerase beta (POLB)) belongs to a member of the DNA polymerase X family, mainly involved in various biological metabolic processes, such as eukaryotic DNA replication, DNA damage repair, gene recombination, and cell cycle regulation. In this study, the muscle development-related gene POLB was screened by selection signature and RNA-seq analysis and then validated for the proliferation and apoptosis of bovine primary myocytes. It was also found that overexpression of the POLB gene had a pro-apoptosis effect, but interfering with the expression of the gene had no significant effect on cells. Then, the analysis of related apoptotic genes revealed that POLB overexpression affected CASP9 gene expression.

Trachoma is the world-leading infectious cause of preventable blindness and is caused by the bacteria Chlamydia trachomatis. In developing countries, diagnosis is usually based on clinical evaluation. Serological-based tests are cheaper than molecular-based ones, but the latter are more sensitive and specific. The present study developed a new duplex qPCR which concomitantly detects the C. trachomatis cryptic plasmid and the human 18S rRNA gene, with an LOD95% for C. trachomatis DNA of 13.04 genome equivalents per reaction. The new qPCR was tested using 50 samples from an endemic area and 12 from a non-endemic area that were previously characterized using direct immunofluorescence assay (DFA) and clinical evaluation. Among the 50 endemic samples, 3 were found to be positive by clinical evaluation (6%), 18 were found to be positive by DFA (36%), and 48 were found to be positive by qPCR (96%). Next, the new duplex qPCR was validated using 50 samples previously characterized by qPCR. Validation was carried out on a benchtop instrument (ABI7500) or on a portable point-of-care instrument (Q3-Plus), showing 95% specificity and 100% sensitivity. The ubiquitous presence of C. trachomatis DNA in samples from the endemic region confirms that constant monitoring is of paramount importance for the effective measurement of the elimination of trachoma. The newly developed duplex qPCR presented in this study, along with its validation in a portable qPCR system, constitutes important tools toward achieving this goal.