Integrating miRNA and full-length transcriptome profiling to elucidate the mechanism of muscle growth in Muscovy ducks reveals key roles for miR-301a-3p/ANKRD1.

BACKGROUND: The popularity of Muscovy ducks is attributed not only to their conformation traits but also to their slightly higher content of breast and leg meat, as well as their stronger-tasting meat compared to that of typical domestic ducks. However, there is a lack of comprehensive systematic research on the development of breast muscle in Muscovy ducks. In addition, since the number of skeletal muscle myofibers is established during the embryonic period, this study conducted a full-length transcriptome sequencing and microRNA sequencing of the breast muscle. Muscovy ducks at four developmental stages, namely Embryonic Day 21 (E21), Embryonic Day 27 (E27), Hatching Day (D0), and Post-hatching Day 7 (D7), were used to isolate total RNA for analysis. RESULTS: A total of 68,161 genes and 472 mature microRNAs were identified. In order to uncover deeper insights into the regulation of mRNA by miRNAs, we conducted an integration of the differentially expressed miRNAs (known as DEMs) with the differentially expressed genes (referred to as DEGs) across various developmental stages. This integration allowed us to make predictions regarding the interactions between miRNAs and mRNA. Through this analysis, we identified a total of 274 DEGs that may serve as potential targets for the 68 DEMs. In the predicted miRNA‒mRNA interaction networks, let-7b, miR-133a-3p, miR-301a-3p, and miR-338-3p were the hub miRNAs. In addition, multiple DEMs also showed predicted target relationships with the DEGs associated with skeletal system development. These identified DEGs and DEMs as well as their predicted interaction networks involved in the regulation of energy homeostasis and muscle development were most likely to play critical roles in facilitating the embryo-to-hatchling transition. A candidate miRNA, miR-301a-3p, exhibited increased expression during the differentiation of satellite cells and was downregulated in the breast muscle tissues of Muscovy ducks at E21 compared to E27. A dual-luciferase reporter assay suggested that the ANKRD1 gene, which encodes a transcription factor, is a direct target of miR-301a-3p. CONCLUSIONS: miR-301a-3p suppressed the posttranscriptional activity of ANKRD1, which is an activator of satellite cell proliferation, as determined with gain- and loss-of-function experiments. miR-301a-3p functions as an inducer of myogenesis by targeting the ANKRD1 gene in Muscovy ducks. These results provide novel insights into the early developmental process of black Muscovy breast muscles and will improve understanding of the underlying molecular mechanisms.

An integrated magnetic separation enzyme-linked colorimetric sensing platform for field detection of Escherichia coli O157: H7 in food.

An intelligent colorimetric sensing platform integrated with in situ immunomagnetic separation function was developed for ultrasensitive detection of Escherichia coli O157: H7 (E. coli O157: H7) in food. Captured antibody modified magnetic nanoparticles (cMNPs) and detection antibody/horseradish peroxidase (HRP) co-functionalized AuNPs (dHAuNPs) were firstly synthesized for targeted enrichment and colorimetric assay of E. coli O157: H7, in which remarkable signal amplification was realized by loading large amounts of HRP on the surface of AuNPs. Coupling with the optical collimation attachments and embedded magnetic separation module, a highly integrated optical device was constructed, by which in situ magnetic separation and high-quality imaging of 96-well microplates containing E. coli O157: H7 was achieved with a smartphone. The concentration of E. coli O157: H7 could be achieved in one-step by performing digital image colorimetric analysis of the obtained image with a custom-designed app. This biosensor possesses high sensitivity (1.63 CFU/mL), short detecting time (3 h), and good anti-interference performance even in real-sample testing. Overall, the developed method is expected to be a novel field detection platform for foodborne pathogens in water and food as well as for the diagnosis of infections due to its portability, ease of operation, and high feasibility.

Smartphone-based microplate reader for high-throughput quantitation of disease markers in serum.

Herein, a smartphone-based portable reader with integrated optics for standard microtiter plates (96 wells) has been designed and demonstrated for high-throughput quantitation of validated biomarkers in serum. The customized optical attachment was simply constructed with a convex lens and a light source, by which the transmitted light through a 96-well microtiter plate was converged for imaging with a smartphone, so that accurate and wide-range reading of the plate can be achieved. More importantly, relying on the digitized colorimetric analysis of the obtained images, concentrations of various biomarkers can be determined directly using the customized mobile app. A set of validated biomarkers for inflammation and infection, C-reactive protein (CRP), serum amyloid A (SAA), and procalcitonin (PCT) have been quantitated with this new system; both the response ranges and limits of detection meet the requirement of clinical tests. The consistency with the results obtained using a commercial microplate reader proves its reliability and precision, augments its potential as a point-of-care diagnostic device for on-site testing or resource-limited settings.

Rapid detection of goose astrovirus genotypes 2 using real-time reverse transcription recombinase polymerase amplification.

BACKGROUND: Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. RESULTS: The real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/µL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. CONCLUSIONS: The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.

Ultrasensitive detection of acephate based on carbon quantum dot-mediated fluorescence inner filter effects.

Acephate is an organophosphorus pesticide (OP) that is widely used to control insects in agricultural fields such as in vegetables and fruits. Toxic OPs can enter human and animal bodies and eventually lead to chronic or acute poisoning. However, traditional enzyme inhibition and colorimetric methods for OPs detection usually require complicated detection procedures and prolonged time and have low detection sensitivity. High-sensitivity monitoring of trace levels of acephate residues is of great significance to food safety and human health. Here, we developed a simple method for ultrasensitive quantitative detection of acephate based on the carbon quantum dot (CQD)-mediated fluorescence inner filter effect (IFE). In this method, the fluorescence from CQDs at 460 nm is quenched by 2,3-diaminophenazine (DAP) and the resulting fluorescence from DAP at 558 nm is through an IFE mechanism between CQDs and DAP, producing ratiometric responses. The ratiometric signal I558/I460 was found to exhibit a linear relationship with the concentration of acephate. The detection limit of this method was 0.052 ppb, which is far lower than the standards for acephate from China and EU in food safety administration. The ratiometric fluorescence sensor was further validated by testing spiked samples of tap water and pear, indicating its great potential for sensitive detection of trace OPs in complex matrixes of real samples.

Quantitative pH Determination Based on the Dominant Wavelength Analysis of Commercial Test Strips.

The determination of pH values is essential in many chemical, medical, and environmental monitoring processes, which has been relying on conventional pH meters (glass electrodes) for quantitation and pH test strips for qualitative (or semi-quantitative) assessment. In this work, we demonstrate a smartphone-based pH determination technique, which performs digital image analysis of commercial test strips, particularly the determination of either the dominant wavelength (λd) or complementary wavelength (λc) of the color image. In conjunction with a 3D-printed optical accessory (with a surface light source and a macro lens), the quality of captured images have been warranted, and the quantitation accuracy of 0.05 pH units has been achieved. More importantly, the performance of this smartphone-based pH reading system (namely "Smart-pH-Reader") was validated using multiple real-world samples, as the results are consistent with those determined with a standard pH meter. The Smart-pH-Reader is envisioned to be a simple, portable, and accurate tool for pH determination in the fields of environmental monitoring, medical diagnosis, and beyond.

IL-35 promotes microglial M2 polarization in a rat model of diabetic neuropathic pain.

Switching microglial polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype represents a novel therapeutic strategy for diabetic neuropathic pain (DNP). This study aims to determine the role and mechanism of interleukin (IL)-35 in regulating microglial M1/M2 polarization in DNP. A rat model of DNP was induced by a single streptozocin injection and recombinant IL-35 (rIL-35) was then intrathecally administered to the rats for 14 days. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to assess the therapeutic effect of IL-35. Highly aggressive proliferating immortalized (HAPI), a rat microglia cell line, was treated with lipopolysaccharide (LPS) for M1 polarization or IL-4 for M2 polarization. The M1 markers (CD68, iNOS, TNF-α, IL-6) and M2 markers (CD206, Arg-1, IL-10) were examined. rIL-35 administration in DNP model rats elevated MWT and TWL, induced microglial polarization toward the M2 phenotype, suppressed JNK signaling and activated JAK2/STAT6 signaling. In vitro assay confirmed that rIL-35 induced microglial M2 polarization in HAPI cells through inhibiting JNK signaling and activating JAK2/STAT6 signaling. Collectively, the mechanism underlying therapeutic effect of IL-35 on DNP may relate to its promotion of microglial M2 polarization by regulating JNK signaling and JAK2/STAT6 signaling.

Eukaryotic expression, Co-IP and MS identify BMPR-1B protein-protein interaction network.

BACKGROUND: BMPR-1B is part of the transforming growth factor ß super family and plays a pivotal role in ewe litter size. Functional loss of exon-8 mutations in the BMPR-1B gene (namely the FecB gene) can increase both the ewe ovulation rate and litter size. RESULTS: This study constructed a eukaryotic expression system, prepared a monoclonal antibody, and characterized BMPR-1B/FecB protein-protein interactions (PPIs). Using Co-immunoprecipitation coupled to mass spectrometry (Co-IP/MS), 23 proteins were identified that specifically interact with FecB in ovary extracts of ewes. Bioinformatics analysis of selected PPIs demonstrated that FecB associated with several other BMPs, primarily via signal transduction in the ovary. FecB and its associated interaction proteins enriched the reproduction process via BMP2 and BMP4 pathways. Signal transduction was identified via Smads proteins and TGF-beta signaling pathway by analyzing the biological processes and pathways. Moreover, other target proteins (GDF5, GDF9, RhoD, and HSP 10) that interact with FecB and that are related to ovulation and litter size in ewes were identified. CONCLUSIONS: In summary, this research identified a novel pathway and insight to explore the PPi network of BMPR-1B.

Prevalence and Antimicrobial Resistance of Salmonella in Pork, Chicken, and Duck from Retail Markets of China.

Salmonella is one of the most important foodborne pathogens associated with animal and human diseases. In this study, 672 samples of fresh meat (pork, 347; chicken, 196; and duck, 129) were collected from retail markets in different provinces of China from 2010 to 2014. We identified 10 different serotypes among 80 Salmonella isolates, whereas 12 isolates were nonmotile precluding conventional identification of complete serotype. Among these 92 isolates, Salmonella enterica serovar Derby (n = 21) was the most prevalent serotype, followed by Salmonella Enteritidis (n = 17), Salmonella Typhimurium (n = 15), Salmonella Indiana (n = 9), Salmonella Agona (n = 7), and Salmonella Assinie (n = 5). Antimicrobial resistance testing for 18 antimicrobial agents revealed that all 92 isolates were resistant to at least 1 antimicrobial agent, and 39 different resistance profiles were identified. The highest resistance was to trimethoprim-sulfamethoxazole (n = 87), followed by tetracycline (n = 51), carbenicillin (n = 38), amoxicillin/A.clav (n = 30), and piperacillin (n = 24). Our results demonstrated that meats presented a potential public health risk, thereby underlining the necessity for local regulatory enforcement agencies in China to monitor salmonellosis.

Design and fabrication of a heterostructured cladding solid-core photonic bandgap fiber for construction of Mach-Zehnder interferometer and high sensitive curvature sensor.

A heterostructured cladding solid-core photonic bandgap fiber (HCSC-PBGF) is designed and fabricated which supports strong core mode and cladding mode transmission in a wide bandgap. An in-line Mach-Zehnder interferometer (MZI) curvature sensor is constructed by splicing single mode fibers at both ends of a HCSC-PBGF. Theoretical analysis of this heterostructured cladding design has been implemented, and the simulation results are consistent with experiment results. Benefiting from the heterostructured cladding design, an enhanced curvature sensing sensitivity of 24.3 nm/m-1 in the range of 0-1.75 m-1 and a high quality interference spectrum with 20 dB fringe visibility are achieved. In order to eliminate the interference of longitudinal strain and transverse torsion on the result of the curvature sensing experiment, we measure the longitudinal strain and transverse torsion sensing properties of HCSC-PBGF, and the results show that the impact is negligible. It is obvious that this high-sensitivity and cost-effective all fiber sensor with a compact structure will have a promising application in fiber sensing.

All fiber M-Z interferometer for high temperature sensing based on a hetero-structured cladding solid-core photonic bandgap fiber.

We proposed and experimentally demonstrated a high temperature fiber sensor using a hetero-structured cladding solid-core photonic bandgap fiber (HCSC-PBGF) for the first time to our knowledge. A hetero-structured cladding solid-core photonic bandgap fiber is designed and fabricated that supports vibrant core mode and cladding mode transmission. Then, an all fiber M-Z interference sensor is constructed by splicing single mode fiber at both ends of HCSC-PBGF without any other micromachining. The transmission characteristics of HCSC-PBGF are analyzed with a full-vector beam propagation method and a full-vector finite element method, and the simulation results are consistent with experiment results. The sensitivity of this fiber sensor is as high as 0.09 nm/°C when operating from room temperature to 1000 °C, and the fringe contrast keeps stable and clear. It is obvious that this all fiber sensor will have great application prospects in fiber sensing with the advantages of a compact structure, high sensitivity, and cost-effectiveness.

Urine multi-index intelligent detection based on polymer/paper hybrid microfluidic biochip for hyperuricemia monitoring.

Hyperuricemia (HUA) has gradually become a public health burden as an independent risk factor for a variety of chronic diseases. Herein, a user-friendly point-of-care (POC) detection system (namely "Smart-HUA-Monitor") based on smartphone-assisted paper-based microfluidic is proposed for colorimetric quantification of HUA urinary markers, including uric acid (UA), creatinine (CR) and pH. The detection limits of UA and CR were 0.0178 and 0.5983 mM, respectively, and the sensitivity of pH were 0.1. The method was successfully validated in artificial urine samples and 100 clinical samples. Bland-Altman plots showed a high consistency between µPAD and the testing instruments (HITACHI 7600 Automatic Analyzer, URIT-500B Urine Analyzer and AU5800B automatic biochemical analyzer) in hospital. Smart-HUA-Monitor provides an accurate quantitative, rapid, low-cost and reliable tool for the monitoring and early diagnosis of HUA urine indicators.

Extracellular matrix-mimetic immunomodulatory fibrous scaffold based on a peony stamens polysaccharide for accelerated wound healing.

Re-establishment of the extracellular matrix (ECM) in wound tissue is critical for activating endogenous tissue repair. In this study, we designed an ECM-like scaffold material using plant polysaccharides and assessed its efficacy through in vitro and in vivo experiments. The scaffold accelerates wound healing by regulating inflammatory responses and accelerating tissue regeneration. Briefly, we isolated two polysaccharides of varying molecular weights from peony stamens. One of the polysaccharides exhibits potent immunomodulatory and tissue regeneration activities. We further prepared electrospinning materials containing this polysaccharide. In vitro investigations have demonstrated the polysaccharide's ability to modulate immune responses by targeting TLR receptors. In vivo experiments utilizing a scaffold composed of this polysaccharide showed accelerated healing of full-thickness skin wounds in mice, promoting rapid tissue regeneration. In conclusion, our study shows that this scaffold can mobilize the endogenous regenerative capacity of tissues to accelerate repair by mimicking the characteristics of ECM. The overall study has implications for the design of new, effective, and safer tissue regeneration strategies.

Detection and genetic diversity of subgroup K avian leukosis virus in local chicken breeds in Jiangxi from 2021 to 2023.

Avian leukosis virus subgroup K (ALV-K) is a new subgroup of avian leukosis virus (ALV) that was first identified in Chinese native chickens in recent years. To further understand the molecular epidemiology and evolutionary diversity of ALV-K, this study investigated the molecular epidemiology of 73,664 chicken plasma samples collected from Jiangxi native chicken flocks. The results showed that ALV-J was the most predominant ALV subtype in Jiangxi native chickens, with a high positivity rate of 4.34%. From 2021 to 2023, there was a gradual upward trend in the proportion of positive numbers of ALV-K among ALV-positive samples, and there was a trend of outbreaks. ALV-J and ALV-K were the main co-infection patterns. Genetic evolutionary analysis based on ALV-K gp85 gene showed that the isolated ALV-K in this study were distributed in various branches of the evolutionary tree with genetic diversity. The homology results showed that the amino acid homology of the isolated ALV-K gp85 gene ranged from 33.9 to 88.1% with the reference strains of subtypes A, B, C, D, E, and J, and from 91.9 to 100% with the other ALV-K reference strains. Multiple mutations were present in the ALV-K gp85, and especially significant mutations were found in the highly variable region hr2. The results of ALV-K replication efficiency showed that the replication efficiency of ALV-K was significantly lower than that of ALV-J. These results enriched the genome sequence data of ALV-K in Chinese geoducks, and laid the foundation for further research on the pathogenesis and prevention of ALV-K.

Alteration of gut microbiome in goslings infected with goose astrovirus.

Goose astrovirus (GoAstV) is an emerging avian pathogen that induces gout in goslings with a mortality of up to 50%. Organ damage caused by GoAstV infection was considered the cause of gout, but it is still unclear whether other factors are involved. Human and murine studies have linked the gut microbiome-derived urate and gout, thus we hypothesized that gut microbiome may also play an important role in gout induced by GoAstV infection. This study tested the pathogenicity of our isolated GoAstV genotype 2 strain on goslings, while the appearance of clinical signs, histopathological changes, viral distribution and the blood level of cytokines were monitored for 18 d postinfection (dpi). The dynamics in the gut microbiome were profiled by 16S sequencing and then correlated with GoAstV infection. Results showed that this study successfully developed an experimental infection model for studying the pathogenicity of the GoAstV infection which induces typical symptoms of gout. GoAstV infection significantly altered the gut microbiome of goslings with the enrichment of potential proinflammatory bacteria and depletion of beneficial bacteria that can produce short-chain fatty acids. More importantly, the microbial pathway involved in urate production was significantly increased in goslings infected with GoAstV, suggesting that gut microbiome-derived urate may also contribute to the gout symptoms. Overall, this study demonstrated the role of gut microbiome in the pathogenesis of GoAstV infection, highlighting the potential of gut microbiome-based therapeutics against gout symptoms.

Heavy metal contamination of urban soils and dusts in Guangzhou, South China.

The heavy metal concentrations of soil and dust samples from roadside, residential areas, parks, campus sport grounds, and commercial sites were studied in Guangzhou, South China. Heavy metals in samples were determined by inductively coupled plasma atomic emission spectrophotometer following acidic digestion with HClO(4) + HF + HNO(3). High concentrations, especially of Cd, Pb, and Zn, were found with mean concentrations of Cd, Cr, Cu, Ni, Pb, and Zn in the urban dusts being 4.22 ± 1.21, 62.2 ± 27.1, 116 ± 30, 31.9 ± 12.6, 72.6 ± 17.9, and 504 ± 191 mg/kg dry weight, respectively. The respective levels in urban soils (0.23 ± 0.19, 22.4 ± 13.8, 41.6 ± 29.4, 11.1 ± 5.3, 65.4 ± 40.2, and 277 ± 214 mg/kg dry weight, respectively), were significantly lower. The integrated pollution index of six metals varied from 0.25 to 3.4 and from 2.5 to 8.4 in urban soils and dusts, respectively, with 61 % of urban soil samples being classified as moderately to highly polluted and all dust samples being classified as highly polluted. The statistical analysis results for the urban dust showed good agreement between principal component analysis and cluster analysis, but distinctly different elemental associations and clustering patterns were observed among heavy metals in the urban soils. The results of multivariate statistic analysis indicated that Cr and Ni concentrations were mainly of natural origin, while Cd, Cu, Pb, and Zn were derived from anthropogenic activities.

GTMNet: a vision transformer with guided transmission map for single remote sensing image dehazing.

Existing dehazing algorithms are not effective for remote sensing images (RSIs) with dense haze, and dehazed results are prone to over-enhancement, color distortion, and artifacts. To tackle these problems, we propose a model GTMNet based on convolutional neural networks (CNNs) and vision transformers (ViTs), combined with dark channel prior (DCP) to achieve good performance. Specifically, a spatial feature transform (SFT) layer is first used to smoothly introduce the guided transmission map (GTM) into the model, improving the ability of the network to estimate haze thickness. A strengthen-operate-subtract (SOS) boosted module is then added to refine the local features of the restored image. The framework of GTMNet is determined by adjusting the input of the SOS boosted module and the position of the SFT layer. On SateHaze1k dataset, we compare GTMNet with several classical dehazing algorithms. The results show that on sub-datasets of Moderate Fog and Thick Fog, the PSNR and SSIM of GTMNet-B are comparable to that of the state-of-the-art model Dehazeformer-L, with only 0.1 times of parameter quantity. In addition, our method is intuitively effective in improving the clarity and the details of dehazed images, which proves the usefulness and significance of using the prior GTM and the SOS boosted module in a single RSI dehazing.

Effect of crossbreeding and sex on slaughter performance and meat quality in Xingguo gray goose based on multiomics data analysis.

Here, we examined the effects of crossbreeding and sex on growth performance, slaughter performance, and meat quality in Xingguo gray (XG) goose, using transcriptomic and metabolomic techniques. The experiment was conducted using 400 goslings (1-day old) of 2 genotypes: the XG breed and its ternary hybrids [F2 geese; (XG Goose♂ × Yangzhou Goose♀)♀ × Shitou Goose♂]. The goslings were divided into 4 groups: female XG, male XG, female F2 geese, and male F2 geese, and growth parameters were examined at 70 d of age, using 30 birds from each group. Following slaughter, samples of breast and thigh muscles were collected from each group for chemical, metabolome, and transcriptome analyses. Growth rate, live body and slaughter weights, meat chemical composition, and muscle fiber diameter were affected by crossbreeding and sex. Crossbreeding significantly improved the dressing percentage, semieviscerated rate, eviscerated yield, and abdominal fat yield of XG geese. To clarify the potential regulatory network affected by crossbreeding and sex, we used RNA-seq and nontargeted metabolomics to detect changes in male and female goose breast muscle. The transcriptome results showed that there were 534, 323, 297, and 492 differently expressed genes (DEGs) among the 4 comparison groups (XG-Female vs. F2-Female, XG-Male vs. F2-Male, F2-Male vs. F2-Female, and XG-Male vs. XG-Female, respectively) that were mainly related to muscle growth and development and fatty acid metabolism pathways. A total of 141 significantly differentially accumulated metabolites (DAMs) were enriched in serine and threonine, propionate, and pyruvate metabolism. Finally, we comprehensively analyzed the metabolome and transcriptome data and found that many DEGs and DAMs played crucial roles in lipid metabolism and muscle growth and development. In summary, crossbreeding can improve XG goose production performance and affect breast muscle gene expression and metabolites in both female and male geese.

Rapid diagnosis of duck Tembusu virus and goose astrovirus with TaqMan-based duplex real-time PCR.

The mixed infection of duck Tembusu virus (DTMUV) and goose astrovirus (GoAstV) is an important problem that endangers the goose industry. Although quantitative PCR has been widely used in monitoring these two viruses, there is no reliable method to detect them at the same time. In this study, by analyzing the published genomes of DTMUV and goose astrovirus genotype 2 (GoAstV-2) isolated in China, we found that both viruses have high conservation, showing 96.5 to 99.5% identities within different strains of DTMUV and GoAstV, respectively. Subsequently, PCR primers and TaqMan probes were designed to identify DTMUV and GoAstV-2, and different fluorescent reporters were given to two probes for differential diagnosis. Through the optimization and verification, this study finally developed a duplex TaqMan qPCR method that can simultaneously detect the above two viruses. The lower limits of detection were 100 copies/µL and 10 copies/µL for DTMUV and GoAstV-2 under optimal condition. The assay was also highly specific in detecting one or two viruses in various combinations in specimens, and provide tool for clinical diagnosis of mixed infections of viruses in goose.