Research Note: Effects of the intermittent feeding of microencapsulation essential oil on laying performance, egg quality, immune response, intestinal morphology, and oxidation status of laying hens.

The aim of this study was to evaluate the effect of microencapsulated essential oils (MEO) on the laying performance, egg quality, immunity, intestinal morphology, and oxidative status of laying hens. A total of 640 Hy-line Brown laying hens, 41 wk of age, were randomly divided into 4 groups, each with 8 replicates containing 20 birds per replicate. The dietary conditions tested included a basal diet (Control) or the basal diet supplemented with various levels of MEO at 100 mg/kg (MEO100), 300 mg/kg (MEO300), and 500 mg/kg (MEO500). The three treatment groups were intermittently fed MEO, following an alternating schedule of 1 wk on and 1 wk off for a total of 56 d. Results showed that feeding MEO at levels of 300 and 500 mg/kg improved both egg production and feed conversion ratios compared to the control group. Hens consumed MEO-supplemented diets exhibited a significant decrease in the breaking egg ratio (P < 0.05) compared to those fed the control diet. Shell thickness and Haugh unit values significantly increased in the groups receiving 300 and 500 mg/kg of MEO (P < 0.05). Both the MEO300 and MEO500 treatments led to improvements in immunoglobulin (IgA, IgM, and IgG) and cytokine (IL-2 and IFN-γ) levels in serum. Hens in the MEO300 and MEO500 groups exhibited higher values for parameters related to intestinal morphometry compared to the control group. Furthermore, supplementation with 300 and 500 mg/kg of MEO enhanced the antioxidant capacity of plasma, as evidenced by increased activities of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and catalase (CAT) (P < 0.05). In summary, the intermittent feeding of MEO improved egg production, enhanced antioxidative processes, immune functions, and intestinal morphology, leading to an amelioration in the egg quality of laying hens. Our data demonstrate that supplementation of 300 mg/kg of MEO in feed can significantly improve animal health and egg quality. Implementation of these feeding practices could have a positive economic impact on poultry and egg industry.

Molecular characterization of a novel mitochondrial NOD-like receptor X1 in chicken that negatively regulates IFN-ß expression via STING.

NOD-like receptor X1 (NLRX1) is known for its unique mitochondrial localization and plays a negative role in innate immunity. The initial characterization and function of chicken NLRX1 remain unclear. Here, chicken mitochondrial-targeted NLRX1 (chNLRX1) protein was identified. It had relatively conserved domains, a unique N-terminal "X" mitochondrial-targeting domain (MT) and 2 highly conserved motifs at positions 510-520 and 412-421. Furthermore, chNLRX1 had a unique 53aa N-terminus-MT consistent with its localization to mitochondria. Additionally, chNLRX1 was observed to reduce the DNA sensing adaptor stimulator of interferon genes (STING)-induced IFN-ß by attenuating the STING-TANK-binding kinase 1 (TBK1) interaction, which is a requisite for the STING-TBK1-IFN-ß signaling pathway. These results suggested that chNLRX1 negatively regulated type-I interferon production via STING in host innate immunity.

In silico and in vitro evaluation of antiviral activity of wogonin against main protease of porcine epidemic diarrhea virus.

The high mortality rate of weaned piglets infected with porcine epidemic diarrhea virus (PEDV) poses a serious threat to the pig industry worldwide, demanding urgent research efforts related to developing effective antiviral drugs to prevent and treat PEDV infection. Small molecules can possibly prevent the spread of infection by targeting specific vital components of the pathogen's genome. Main protease (Mpro, also named 3CL protease) plays essential roles in PEDV replication and has emerged as a promising target for the inhibition of PEDV. In this study, wogonin exhibited antiviral activity against a PEDV variant isolate, interacting with the PEDV particles and inhibiting the internalization, replication and release of PEDV. The molecular docking model indicated that wogonin was firmly embedded in the groove of the active pocket of Mpro. Furthermore, the interaction between wogonin and Mpro was validated in silico via microscale thermophoresis and surface plasmon resonance analyses. In addition, the results of a fluorescence resonance energy transfer (FRET) assay indicated that wogonin exerted an inhibitory effect on Mpro. These findings provide useful insights into the antiviral activities of wogonin, which could support future research into anti-PEDV drugs.`.

Effects of Compound Probiotics on Cecal Microbiota and Metabolome of Swine.

Complex probiotics are made from various single probiotics mixed in scientific formula. The long-term intake of different probiotics is beneficial to maintain the intestinal microecological balance, inhibiting harmful pathogenic flora and facilitating organism health. Based on the limited research on intestinal flora and related metabolites after the long-term intake of the probiotic complex, in this study, 16S rRNA gene sequencing and untargeted metabolomics were used to further investigate the effects of the probiotic complex on the intestinal flora and metabolome of pigs. The results demonstrated that the content of flora in the intestinal tract or metabolites of pigs varied greatly and was related to cellular metabolic pathways after the long-term feeding of complex probiotics. This study provides a valuable theoretical basis for farmers to raise pigs scientifically and healthily.

TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of GoAstV, GPV, and GoCV.

Goose astrovirus (GoAstV), goose parvovirus (GPV), and goose circovirus (GoCV) infections have similar symptoms, such as severe diarrhea, and cause serious economic losses to the goose industry globally. Therefore, it is necessary to develop a rapid and accurate method for the differential diagnosis of the 3 viruses. In this study, a TaqMan probe-based multiplex reverse transcription-qualitative polymerase chain reaction (RT-qPCR) method was established and optimized for simultaneous detection of the three viruses. Three pairs of specific primers and probes were designed considering the conserved sequences of ORF2, VP3, and Rep of GoAstV, GPV, and GoCV, respectively. Singleplex real-time RT-qPCR detected a minimum of 10 copies of these genes, while multiplex real-time RT-qPCR detected a minimum of 100 copies. The correlation coefficients exceeded 0.99, and the amplification efficiency was 80 to 100%. The assay had high sensitivity, specificity, and repeatability. In 85 tissue samples, GoAstV and GPV were the main pathogens and demonstrated co-infection. This assay provides a rapid, efficient, specific, and sensitive tool for the detection of GoAstV, GPV, and GoCV. This can facilitate disease management and epidemiological surveillance.

Chicken anemia virus VP1 negatively regulates type I interferon via targeting interferon regulatory factor 7 of the DNA-sensing pathway.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays a vital role in sensing viral DNA in the cytosol, stimulating type I interferon (IFN) production and triggering the innate immune response against DNA virus infection. However, viruses have evolved effective inhibitors to impede this sensing pathway. Chicken anemia virus (CAV), a nonenveloped ssDNA virus, is a ubiquitous pathogen causing great economic losses to the poultry industry globally. CAV infection is reported to downregulate type I IFN induction. However, whether the cGAS-STING signal axis is used by CAV to regulate type I IFN remains unclear. Our results demonstrate that CAV infection significantly elevates the expression of cGAS and STING at the mRNA level, whereas IFN-ß levels are reduced. Furthermore, IFN-ß activation was completely blocked by the structural protein VP1 of CAV in interferon stimulatory DNA (ISD) or STING-stimulated cells. VP1 was further confirmed as an inhibitor by interacting with interferon regulatory factor 7 (IRF7) by binding its C-terminal 143-492 aa region. IRF7 dimerization induced by TANK binding kinase 1 (TBK1) could be inhibited by VP1 in a dose-dependent manner. Together, our study demonstrates that CAV VP1 is an effective inhibitor that interacts with IRF7 and antagonizes cGAS-STING pathway-mediated IFN-ß activation. These findings reveal a new mechanism of immune evasion by CAV.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of four feline diarrhea-associated viruses.

Since their recent discovery, the prevalence of novel feline enteric viruses, including feline bocavirus 1 (FBoV-1), feline astrovirus (FeAstV), and feline kobuvirus (FeKoV), has been reported in China. Co-infections of these viruses with feline parvovirus (FPV) are common causes of diarrhea in cats. Viral co-infections are difficult to identify because of their non-specific clinical signs. To detect and identify these viruses, a quick and specific pathogen-testing approach is required. Here, we establish a real-time PCR (qPCR) based on multiple TaqMan probes for the simultaneous detection of FBoV-1, FeAstV, FeKoV, and FPV. Specific primers and TaqMan fluorescent probes were designed to ensure specificity. The results showed that the detection limit of single qPCR was up to 10 copies, and the detection limit of multiplex qPCR was up to 100 copies, with correlation coefficients >0.995 in all cases. Clinical sample detection revealed a 25.19% (34/135) total rate of co-infection among the viruses and a 1.48% (2/135) quadruple infection rate. Thus, this multiplex qPCR approach can serve as a quick, sensitive, and specific diagnostic tool for FBoV-1, FeAstV, FeKoV, and FPV identification, and it may be utilized for routine surveillance of these emerging and reemerging feline enteric viruses.

Establishment of TaqMan-based real-time PCR assay for rapid detection of duck circovirus.

Duck circovirus (DuCV) is widespread across the world and causes feather disorders in young ducks. It was identified as the causative pathogen of duck beak atrophy and dwarfism syndrome and primary sclerosing cholangitis. In this study, we aimed to establish a TaqMan-based real-time PCR assay to detect DuCV. The primers and probe were designed based on the conserved region of the DuCV Rep gene. After optimizing the reaction conditions, the minimum virus detection limit of the designed PCR technique was 39.4 copies/µL, 100 times that of conventional PCR (cPCR). No cross-reaction with six other common duck viruses was observed. The intra- and inter-assay variations were less than 1%. The detection rate of DuCV-positive clinical samples using TaqMan-based real-time PCR was higher than that using SYBR Green-based real-time PCR and cPCR. Collectively, these results showed that the established TaqMan-based real-time PCR detected DuCV with high sensitivity and specificity, and significant repeatability, making it suitable for clinical use. Hence, it may be used as a novel tool for the diagnosis and epidemiological investigation of DuCV.

A duplex SYBR green I-based real-time polymerase chain reaction assay for concurrent detection of feline parvovirus and feline coronavirus.

Feline coronavirus (FCoV) contains two serotypes, feline enteric coronavirus (FECV) and Feline infectious peritonitis virus (FIPV). FECV and feline parvovirus (FPV) can cause similar clinical symptoms in cats, such as diarrhea. The objective of this study was to establish a duplex SYBR Green I-based quantitative polymerase chain reaction (qPCR) assay for rapid and simultaneous detection of FPV and FCoV. Two pairs of specific PCR primers were designed to target fragments of the VP2 gene of FPV and of the 5' UTR gene of FCoV, respectively. The assay distinguished between the two viruses based on the melting curves (melting temperatures 77.0 ± 0.5 °C [FPV] and 80.5 ± 0.5 °C [FCoV]). The minimum limits of FPV and FCoV detection were 4.74 × 101 copies/µL and 7.77 × 101 copies/µL, respectively. The assay showed excellent reproducibility and reliability, based on the mean coefficient of variation. In conclusion, this novel duplex SYBR Green I-based qPCR assay is sensitive and can specifically, reliably, and rapidly detect FPV and FCoV (co-)infections.

Simultaneous detection of feline parvovirus and feline bocavirus using SYBR Green I-based duplex real-time polymerase chain reaction.

Since both feline parvovirus (FPV) and feline bocavirus (FBoV) can cause diarrhea in cats, it is difficult to distinguish them clinically. This study aimed to develop a SYBR Green I-based duplex real-time polymerase chain reaction (PCR) assay for distinguishing FPV and FBoV-1 on the basis of the melting temperature of the PCR product. A total of 132 fecal samples from different domestic and feral cats were collected, and the results of SYBR Green I-based duplex real-time PCR assay were compared with those of the traditional PCR assay for a comprehensive evaluation. The melting temperatures were found to be 86 °C and 77.5 °C for FBoV-1 and FPV, respectively, and no specific melting peaks for other non-targeted feline viruses were observed. The data obtained from this assay had a good linear relationship; the detection limits of FPV and FBoV-1 were 2.907 × 101 copies/µL and 3.836 × 101 copies/µL, respectively. In addition, the experiment exhibited high reproducibility. The positive detection rates of the SYBR Green I-based duplex real-time PCR assay for FPV and FBoV-1 were 16.67% (22/132) and 6.82% (9/132), respectively, and the positive detection rate for co-infection with FPV and FBoV-1 was 3.03% (4/132). This result was much more sensitive than that of the traditional PCR method. Thus, the developed SYBR Green I-based assay is a sensitive, rapid, specific, and reliable method for the clinical diagnosis of FPV and FBoV-1 and can provide technical support for the simultaneous detection of co-infection with these viruses in the future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02947-w.

Development of a TaqMan-based real-time assay for the specific detection of canine astrovirus.

In this study, a TaqMan-based real-time polymerase chain reaction (PCR) method to detect canine astrovirus in clinical samples was developed. Primers and probes were designed to target conserved regions of the complete viral genome sequence. The results showed that the proposed method can detect a minimum of 101 copy numbers. No cross-reactivity with other canine and feline viruses was observed. The coefficient of variation was <5%. Evaluation of the clinical samples showed that quantitative PCR had a 5.26 % higher positive detection rate than conventional PCR. These results indicate that the method developed in this study is highly reliable and suitable for veterinary clinical diagnosis and epidemiological investigations.

Phylogenetic analysis and evolution of feline bocavirus in Anhui Province, eastern China.

To understand the epidemic status of feline bocavirus (FBoV) in Anhui Province, eastern China, FBoV was successfully extracted from fecal samples of domestic cats, and five complete genomes were amplified in this study. Phylogenetic analysis showed that these five strains belong to three different FBoV genotypes. Recombination analysis showed that inter- and intra-genotype recombination events occurred. Selection pressure and codon usage bias analyses indicated that FBoV-1 and FBoV-3 continuously evolve toward adaptation, and selection pressure is the main factor for codon usage bias during evolution. This study provides the first molecular evidence of FBoV prevalence in eastern China, further enriching the available information on its genetics and evolutionary characteristics and providing a basis for further research on its evolution.

Effect of fermented biogas residue on growth performance, serum biochemical parameters, and meat quality in pigs.

OBJECTIVE: This study investigated the effect of fermented biogas residue (FBR) of wheat on the performance, serum biochemical parameters, and meat quality in pigs. METHODS: We selected 128 pigs (the mean initial body weight was 40.24±3.08 kg) and randomly allocated them to 4 groups (1 control group and 3 treatment groups) with 4 replicates per group and 8 pigs per pen in a randomized complete block design based on initial body weight and sex. The control group received a corn-soybean meal-based diet, the treatment group fed diets containing 5%, 10%, and 15% FBR, respectively (abbreviated as FBR5, FBR10, and FBR15, respectively). Every group received equivalent-energy and nitrogen diets. The test lasted 60 days and was divided into early and late stages. Blood and carcass samples were obtained on 60 d. Meat quality was collected from two pigs per pen. RESULTS: During the late stage, the average daily feed intake and average daily gain of the treatment groups was greater than that of the control group (p<0.05). During the entire experiment, the average daily gain of the treatment groups was higher than that of the control group (p<0.05). Fermented biomass residue did not significantly affect serum biochemical parameters or meat quality, but did affect amino acid profiles in pork. The contents of Asp, Arg, Tyr, Phe, Leu, Thr, Ser, Lys, Pro, Ala, essential amino acids, non-essential amino acids, and total amino acids in pork of FBR5 and FBR10 were greater than those of the control group (p<0.05). CONCLUSION: These combined results suggest that feeding FBR could increase the average daily gain and average daily feed intake in pigs and the content of several flavor-promoting amino acids.

Avian leukosis virus subgroup J triggers caspase-1-mediated inflammatory response in chick livers.

Many pathogens trigger caspase-1-mediated innate immune responses. Avian leukosis virus subgroup J (ALV-J) causes serious immunosuppression and diverse tumors in chicks. The caspase-1 inflammasome mechanism of response to ALV-J invading remains unclear. Here we investigated the expression of caspase-1, the inflammasome adaptor NLRP3, IL-1ß and IL-18 in response to ALV-J infection in the liver of chick. We found caspase-1 mRNA expression was elevated at 5 dpi and peaked at 7 dpi in ALV-J infected animals. Corresponding to this, the expressions of NLRP3 and proinflammatory cytokines IL-1ß and IL-18 were significantly increased at 5 or 7 dpi. In addition, caspase-1 protein expression and inflammatory cell infiltration were induced after virus infection. These results indicated that ALV-J infection could trigger the caspase-1- mediated inflammatory response in chicks. Thus, an understanding of the inflammatory responses can provide a better insight into the pathogenicity of ALV-J and a possible anti-virus target for ALV-J infection.

The efficacy of chimeric vaccines constructed with PEP-1 and Ii-Key linking to a hybrid epitope from heterologous viruses.

The heterologous epitope-peptide from different viruses may represent an attractive candidate vaccine. In order to evaluate the role of cell-permeable peptide (PEP-1) and Ii-Key moiety from the invariant chain (Ii) of MHC on the heterologous peptide chimeras, we linked the two vehicles to hybrid epitopes on the VP2 protein (aa197-209) of the infectious bursal disease virus and HN protein (aa345-353) of the Newcastle disease virus. The chimeric vaccines were prepared and injected into mice. The immune effects were measured by indirect ELISA. The results showed that the vehicle(s) could significantly boost immune effects against the heterologous epitope peptide. The Ii-Key-only carrier induced more effective immunological responses, compared with the PEP-1 and Ii-Key hybrid vehicle. The carrier-peptide hybrids all showed strong colocalization with major histocompatibility complex (MHC) class II molecules compared with the epitope-peptide (weakly-binding) after co-transfection into 293T cells. Together, our results lay the groundwork for designing new hybrid vaccines based on Ii-Key and/or PEP-1 peptides.

The intracellular localization and association of porcine Ia-associated invariant chain with the MHC class I-related porcine neonatal Fc receptor for IgG.

The neonatal Fc receptor (FcRn) transports IgG from mother to young and is involved in antigen presentation. FcRn is structurally similar to MHC class I, but its intracellular trafficking pathway is much more analogous to that of MHC class II. Ia-associated invariant chain (Ii) molecules play an additional role in directing MHC class II trafficking within the endocytic compartments by physical association with MHC class II. This study addresses the question of whether pig Ii chain plays this important role in FcRn trafficking to the endoplasmic reticulum. Red or green fluorescent protein-fused Ii or FcRn was constructed, and the intracellular localization of pig Ii with FcRn was detected using confocal microscopy. Immunoprecipitation and western blotting were used to test for their association. The results indicate that pig Ii chain specifically interacts with both FcRn H chain alone and FcRn-ß2m complex, and the CLIP in Ii was required for FcRn-Ii association. A truncated FcRn deletion in the cytoplasmic tail changed the intracellular localization of FcRn. However, the truncated FcRn can still combine Ii. This indicated that the cytoplasmic tail of FcRn fails to affect FcRn association with Ii. These results suggest that association of FcRn with Ii chain is relevant, and appreciation of this process is important to the understanding of how IgG is transported.

[Isolation and identification of thermophilic bacteria for efficient dead-pig composting].

OBJECTIVE: To isolate thermophilic bacteria to degrade organic substances of dead-pig. METHODS: Primary screening was done by using diluted plate count and selective medium, and then enzyme activity was measured for secondary screening. Two thermophilic bacterial strains N-3 and Y-3 were isolated, and could degrade protein and lipids. To test their effect, the isolates were mixed (V: V = 1:1, the number of bacteria was 10(8) CFU/mL) and inoculated in dead-pigs and sawdust composting with different doses (0%, 0.3%, 0.6% and 0.9% of the wet weight of fermentation materials). RESULTS: Strain N-3 was identified as Bacillus aestuarii and Y-3 as Geobacillus thermodenitrificans, based on their 16S rDNA gene sequences. The composting temperature of the 0.3%, 0.6% and 0.9% inoculation group could reach 60 degrees C and maintain at the high temperature for about 10 d, which is higher than control (P < 0.01). At the end of composting, the dead-pig degradation rate of the (0%, 0.3%, 0.6% and 0.9% inoculation groups were 71.2%, 75.7%, 96.7% and 97.1%, respectively. The groups of 0.6% and 0.9% were significantly higher than the control (P < 0.01). CONCLUSION: Sufficient amount inoculation of thermophilic bacteria (> 0.6%) could effectively increase composting temperature, maintain thermophilic stage for longer time, and accelerate degradation of dead-pig by composting.

[Effect of the targeted mutation of sorting motifs on the localization of porcine Ia-associated invariant chain].

OBJECTIVE: To investigate the effect of amino acids around two Leu-based motifs (Leu 7/Ile 8 and Met 16/Leu 17) in the cytosolic tail of porcine Ia-associated invariant chain (Ii) on endomembrane system positioning function. METHODS: At first, two Leu-based motifs and amino acids around motifs were mutated through site-directed mutagenesis of the megaprimer PCR method, and the mutant genes were ligated to the vector pEGFP-C1, respectively. Twenty-one mutations of Ii recombinant plasmid were obtained and transiently transfected into the COS-7 cells by Lipofectamine(TM) 2000. Fluorescence microscopy was used to detect the intracellular localization of mutation Ii. RESULTS: Leu 7/Ile 8 and Met 16/Leu 17 independently mediated the intracellular localization of mutation Ii. When one leucine motif was kept and the amino acid around the other leucine motif was mutated, fusion protein GFP-Ii was located in the endomembrane system or distributed in the whole cell. CONCLUSION: pig Ii comprises two independent sorting signals, and the functional Leu-based sorting signal requires specific neighboring residues.

Cross-species association of quail invariant chain with chicken and mouse MHC II molecules.

There are different degrees of similarity among vertebrate invariant chains (Ii). The aim of this study was to determine the relationship between quail and other vertebrate Ii MHC class II molecules. The two quail Ii isoforms (qIi-1, qIi-2) were cloned by RACE, and qRT-PCR analysis of different organs showed that their expression levels were positively correlated with MHC II gene (B-LB) transcription levels. Confocal microscopy indicated that quail full-length Ii co-localized with MHC II of quail, chicken or mouse in 293FT cells co-transfected with both genes. Immunoprecipitation and western blotting further indicated that these aggregates corresponded to polymers of Ii and MHC class II molecules. This cross-species molecular association of quail Ii with chicken and mouse MHC II suggests that Ii molecules have a high structural and functional similarity and may thereby be used as potential immune carriers across species.

Boosting immune response with the invariant chain segments via association with non-peptide binding region of major histocompatibility complex class II molecules.

BACKGROUND: Based on binding of invariant chain (Ii) to major histocompatibility complex (MHC) class II molecules to form complexes, Ii-segment hybrids, Ii-key structure linking an epitope, or Ii class II-associated invariant chain peptide (CLIP) replaced with an epitope were used to increase immune response. It is currently unknown whether the Ii-segment cytosolic and transmembrane domains bind to the MHC non-peptide binding region (PBR) and consequently influence immune response. To investigate the potential role of Ii-segments in the immune response via MHC II/peptide complexes, a few hybrids containing Ii-segments and a multiepitope (F306) from Newcastle disease virus fusion protein (F) were constructed, and their binding effects on MHC II molecules and specific antibody production were compared using confocal microscopy, immunoprecipitation, western blotting and animal experiments. RESULTS: One of the Ii-segment/F306 hybrids, containing ND (Asn-Asp) outside the F306 in the Ii-key structure (Ii-key/F306/ND), neither co-localized with MHC II molecules on plasma membrane nor bound to MHC II molecules to form complexes. However, stimulation of mice with the structure produced 4-fold higher antibody titers compared with F306 alone. The two other Ii-segment/F306 hybrids, in which the transmembrane and cytosolic domains of Ii were linked to this structure (Cyt/TM/Ii-key/F306/ND), partially co-localized on plasma membrane with MHC class II molecules and weakly bound MHC II molecules to form complexes. They induced mice to produce approximately 9-fold higher antibody titers compared with F306 alone. Furthermore, an Ii/F306 hybrid (F306 substituting CLIP) co-localized well with MHC II molecules on the membrane to form complexes, although it increased antibody titer about 3-fold relative to F306 alone. CONCLUSIONS: These results suggest that Ii-segments improve specific immune response by binding to the non-PBR on MHC class II molecules and enabling membrane co-localization with MHC II molecules, resulting in the formation of relatively stable MHC II/peptide complexes on the plasma membrane, and signal transduction.