Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. The capsule is an important virulence determinant of many pathogenic bacteria, but the function of the capsule in Av. paragallinarum is not well defined. In this study, acapsular mutants of Av. paragallinarum were constructed by inactivation of the hctA gene using the TargeTron gene knockout system. The acapsular mutants were found to have greater hemagglutination activity than did the wild-type strain. Further, acapsular mutants exhibited an increased ability to adhere to DF-1 cells and to form biofilms on abiotic surfaces. Virulence assays showed that acapsular mutants were less virulent than the wild-type strain. Taken together, these results indicated that loss of capsule increases hemagglutination and adhesion activities but decreases the virulence of Av. paragallinarum. These results could be valuable to further elucidate the function of the capsule and the mechanism of pathogenicity of Av. paragallinarum.

Regulation of coronaviral poly(A) tail length during infection is not coronavirus species- or host cell-specific.

It has been demonstrated that the length of the poly(A) tail in the bovine coronavirus (BCoV), which belongs to genus betacoronaviruses, is regulated throughout infection in human rectal tumor-18 (HRT-18) cells, and the length of the poly(A) tail is associated with the efficiency of virus translation. Here, we examined whether the regulation of viral poly(A) tail length is cell-type independent and whether it is a common feature of coronaviruses to assess the significance of the regulation. By ligating head-to-tail viral RNA positive strands and sequencing, we found that (1) the regulation pattern of coronaviral poly(A) tail length in BCoV-infected hamster kidney-21 (BHK-21) cells was similar to that in BCoV-infected HRT-18 cells and (2) the poly(A) tail length of wild-type avian infectious bronchitis virus (IBV) virulent strain IBV-TW1, which is in the genus gammacoronaviruses, varied throughout infection in primary chicken embryo kidney (CEK) cells and in the tracheas of 1-day-old chicks. Interestingly, the poly(A) tail length variation was similarly found in the avirulent IBV strain H120 in CEK cells, although the overall poly(A) tail length was shorter for this virus. The results suggest that the regulation of coronaviral poly(A) tail length during infection may be a common feature among coronaviruses and can occur in a noncancerous cell line (BHK-21 cells), primary cell culture (CEK cells), and living system (chickens), further reinforcing the biological significance of this regulation during coronavirus infection.

Improved nonaqueous capillary electrophoresis for tetracyclines at subparts per billion level.

A NACE method with laser-induced fluorescence detection was modified for sensitive detection of 4 tetracyclines (TCs) in biological samples and feeds. The changes in injection mode, injection times, id of capillary, excitation wavelength, and the use of surfactant and sample stacking technique all contributed to improved LODs of TCs to sub-ng/mL level. With the optimized conditions, the instrumental LODs could reach 1.33 ng/mL for chlorotetracycline (CTC) and 13.3 ng/mL for TC, oxytetracycline (OTC), and doxycycline (DC), an improvement of 10-100-fold over past studies. A simple SPE procedure was further developed for the extraction and concentration of TCs in plasma, urine, feed, and milk. Taken together, the instrumental LOD and feasible SPE concentration factors the overall LODs for CTC could reach 65 pg/mL in feed and milk and 260 pg/mL in plasma and urine. Detection limits for TC, OTC, and DC at sub-ng/mL level were also achieved. The modified CE-LIF method was found to be less complicated and more sensitive than the best current methods using UV or LIF detection, and has been applied successfully to assess oral absorption of DC in swine and chickens and to confirm suspected TC-positive bovine serum samples.

Application of purified recombinant antigenic spike fragments to the diagnosis of avian infectious bronchitis virus infection.

The spike (S) protein, containing two subunits, S1 and S2, is the major immunity-eliciting antigen of avian infectious bronchitis virus (IBV), a highly contagious disease of chickens. Several immunogenic regions, mainly located within the S1 subunit, have been identified. Nonetheless, these immune-dominant regions were defined using selected monoclonal antibodies or using a short peptide approach that involves only certain limited regions of the S protein. In addition, some immune-dominant regions are located in hypervariable regions (HVRs) which are not present in all serotypes. Hence, the aim of this study was to determine a broader range of antigenic regions that have strong antibody eliciting ability; these could then be applied for development of an IBV-diagnostic tool. Initially, the S1 and part of the S2 subunit protein (24-567 amino acids) were expressed as five fragments in prokaryotic system. The antigenicity was confirmed using IBV immunized sera. Performance of the S subfragments was evaluated by ELISA using a panel of field chicken sera with known IBV titres determined by a commercial kit. This indicated that, among the five antigenic recombinant proteins, the region S-E showed the highest specificity and sensitivity, namely 95.38 % and 96.29 %, respectively. The κ value for the in-house ELISA using the S-E fragment compared to a commercial kit was 0.9172, indicating a high agreement between these two methods. As region S-E harbors strong immunogenicity within the spike protein, it has the potential to be exploited as an antigen when developing a cost-effective ELISA-based diagnosis tool.

Identification and characterization of an RTX toxin-like gene and its operon from Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Whole-genome sequencing analysis showed that A. paragallinarum strain H18 contains an RTX toxin-like operon with strong similarity to the RTX operons of other members of the Pasteurellaceae. Four genes, designated avxIC, avxIA, avxIB, and avxID, were found in this operon. The avxIA gene encodes the structural RTX toxin-like protein, which has a predicted molecular mass of about 250 kDa. The AvxIA protein contains a peptidase S8 domain and a proprotein convertase P-domain, neither of which has been found in other RTX toxins. Recombinant AvxIA proteins expressed in Escherichia coli showed neither hemolytic nor cytotoxic activity. Polymerase chain reaction and sequencing analysis revealed that the avxIA gene was present in all strains and field isolates of A. paragallinarum examined in this study. Sera collected from chickens exposed to A. paragallinarum exhibited strong reactivity to the AvxIA protein, which suggests that AvxIA is immunogenic. This is the first report of the identification of an RTX toxin-like operon from A. paragallinarum. The gene products of this operon may be related to disease pathogenesis and potentially represent a useful vaccine target of A. paragallinarum.

Molecular Characterization and Pathogenicity of the Novel Recombinant Muscovy Duck Parvovirus Isolated from Geese.

Goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) are the main agents associated with waterfowl parvovirus infections that caused great economic losses in the waterfowl industry. In 2020, a recombinant waterfowl parvovirus, 20-0910G, was isolated in a goose flock in Taiwan that experienced high morbidity and mortality. The whole genome of 20-0910G was sequenced to investigate the genomic characteristics of this isolate. Recombination analysis revealed that, like Chinese rMDPVs, 20-0910G had a classical MDPV genomic backbone and underwent two recombination events with classical GPVs at the P9 promoter and partial VP3 gene regions. Phylogenetic analysis of the genomic sequence found that this goose-origin parvovirus was highly similar to the circulating recombinant MDPVs (rMDPVs) isolated from duck flocks in China. The results of experimental challenge tests showed that 20-0910G caused 100% mortality in goose embryos and in 1-day-old goslings by 11 and 12 days post-inoculation, respectively. Taken together, the results indicated that this goose-origin rMDPV was closely related to the duck-origin rMDPVs and was highly pathogenic to young geese.

Prevalence and Genotyping of Chlamydia psittaci from Domestic Waterfowl, Companion Birds, and Wild Birds in Taiwan.

Chlamydia psittaci, the causative agent of avian chlamydiosis, an important zoonotic disease, infects a wide range of birds. Infected birds, whether symptomatic or asymptomatic, intermittently shed the agent through respiratory and intestinal routes. Therefore, it is essential to investigate the epizootiology of C. psittaci in poultry, pet birds, and wild birds. In this study, cloacal or fecal swabs collected from domestic waterfowl, psittacine birds, Columbidae, and wild birds were used to determine the prevalence of C. psittaci in Taiwan between 2014 and 2017. The C. psittaci infection rate was as high as 34.2% among domestic waterfowl farms. The waterfowl isolates clustered into two groups based on ompA phylogeny: one group (G1-like) clustered with the Polish G1 strains; the other group (waterfowl-TW) clustered near, but independently from, the classical ABE genotype cluster. Separately, 3.1% of parrot samples tested positive for C. psittaci belonging to genotype A. C. psittaci isolates of genotype B were detected in 10.1% of racing pigeons and other Columbidae. Wild bird samples from a wildlife refuge had a 2.2% prevalence rate; among these, two atypical C. psittaci genotypes were detected in samples from a Malayan night heron (Gorsachius melanolophus) and a Taiwan barbet (Megalaima nuchalis). Taken together, our results revealed that the risk of C. psittaci transmission from domestic waterfowl and Columbidae birds to humans could be underestimated, given the high prevalence rates in these birds. Furthermore, the free-range rearing system of waterfowl in Taiwan may promote C. psittaci transmission between poultry and wild birds. Pet birds and racing pigeons, which are in close contact with people, are also possible sources for cross-species transmission. Further studies are necessary to elucidate the virulence, biological and genetic characteristics, and modes of transmission of Taiwanese C. psittaci isolates to facilitate the prevention and control of C. psittaci infection.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

Development of an enzyme-linked immunosorbent assay for detecting infectious bursal disease virus (IBDV) infection based on the VP3 structural protein.

Infectious bursal disease virus (IBDV) causes a contagious immunosuppressive disease in chickens. The aim of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using the expressed VP2 or VP3 protein of IBDV as the coating antigen for detecting antibodies to IBDV. Experimental results were compared with virus neutralization assay and a commercial-available ELISA. These assays were used to examine the sera from farm chickens and chickens vaccinated experimentally. The VP3-based ELISA had a higher correlation coefficient (R2) of 0.812 with a commercial ELISA kit at a serum dilution of 1:500 than that of VP2-based ELISA (R2) of 0.671. The relative sensitivity between virus neutralization and VP2-ELISA and VP3-ELISA was 96% (251/262) and 100% (262/262), respectively, and that between virus neutralization and a commercial ELISA was 99% (257/261). Additionally, compared with virus neutralization assay, the reference technique for diagnosing IBDV, VP3-based ELISA had an agreement value of 99%, superior to that of VP2-based ELISA (95%) or the commercial kit (89%). These results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial one, which is generally used to replace the virus neutralization assay. However, the preparation of VP3 is derived from an Escherichia coli expression system with a high yield and purification efficiency by Ni2+-NTA gels, which is more favorable to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostic method for IBDV infection in field chickens.

Hyperlipidemia in rabbit hemorrhagic disease.

Clinically healthy rabbits were inoculated with rabbit hemorrhagic disease virus (RHDV) and the kinetics of their serum lipid parameters and liver enzymes were monitored. After RHDV inoculation, hyperlipidemia was observed (P(triglyceride)<0.0001, P(cholesterol)=0.0003) along with significant increases in serum aspartate aminotransferase, alanine aminotransferase and gamma-glutamyltransferase (P<0.0001). An exponential increase in serum triglyceride was also seen. Thus, the presence of hyperlipidemia (from 30 h post-inoculation) in the infected rabbits points to impairment in lipid metabolism. This is the first demonstration that RHDV infection leads to hyperlipidemia, probably due to the disorder of liver enzymes associated with lipid metabolism.

Impairment of renal function and electrolyte balance in rabbit hemorrhagic disease.

Rabbit hemorrhagic disease virus (RHDV) induced viral fulminant hepatitis in adult rabbits. We investigated the damage of renal function and electrolyte balance in experimentally infected rabbit by measuring the related serum parameters to elucidate the pathogenesis of RHDV as an index for medical treatment. Nineteen New Zealand White rabbits, ten females and nine males, were each intramuscularly inoculated with 0.5 ml 50% rabbit lethal dose (RLD(50)) rabbit hemorrhagic disease virus. Blood samples were collected at 0 hr post inoculation (HPI) and every 6 hr from 18 HPI repeatedly through 66 HPI. After virus inoculation, serum blood urea nitrogen (BUN), creatinine (CREA) and sodium (Na(+)) were elevated to a highly significant level (p<0.0001), whereas serum potassium (K(+)) was moderately elevated to a significant level (p<0.05). Hypoglycemia developed highly significantly (p<0.0001). Serum chloride ion (Cl(-)) was the only parameter which did not change significantly (p=0.077). No significant sexual difference was observed among these parameters. Renal insufficiency progressed from 36 hr, as indicated by the increases in BUN and CREA; significant changes in electrolytes resulting in the increased osmolality of extracellular fluid that induced flow disturbance which consequently destroy the homeostasis in cells. Therefore, the later impairments in renal function and electrolyte balance might be an important threat for rabbits which might have survived from acute fulminant hepatitis in RHD.

Cloning of the gene and characterization of the enzymatic properties of the monomeric alkaline phosphatase (PhoX) from Pasteurella multocida strain X-73.

We have identified a new phoX gene encoding the monomeric alkaline phosphatase from Pasteurella multocida X-73. This gene was not found in the published genome sequence of Pasteurella multocida pm70. Characterization of the recombinant PhoX of Pasteurella multocida X-73 showed that it is a monomeric enzyme, activated by Ca(2+) and possibly secreted by the Tat pathway. These features distinguish phosphatases of the PhoX family from those of the PhoA family. All proteins of the PhoX family were found to contain a conserved motif that shares significant sequence homology with the calcium-binding site of a phosphotriesterase known as diisopropylfluorophosphatase. Site-directed mutagenesis revealed that D527 of PhoX might be the ligand bound to the catalytic calcium. This is the first report on identification of homologous sequences between PhoX and the phosphotriesterase and on the potential calcium-binding site of PhoX.

Imaging, biodistribution and efficacy evaluation of 188Re-human serum albumin microspheres via intraarterial route in an orthotopic hepatoma model.

PURPOSE: Liver cancer is the second most common cause of death worldwide. This study was to investigate the SPECT/CT, ultrasound, biodistribution and therapeutic evaluation of 188Re-human serum albumin microspheres (188Re-HSAM) in the GP7TB orthotopic hepatoma rat model. MATERIALS AND METHODS: HSAM was labeled with 188Re by using a home-made kit and microwave system. The 188Re-HSAM was administered via intraarterial route. The in vivo distribution of 188Re-HSAM was determined by biodistribution analysis and nanoSPECT/CT system. In efficacy, tumor volumes were tracked longitudinally by three-dimensional ultrasound. RESULTS: The biodistribution and nanoSPECT/CT imaging showed that 188Re-HSAM could accumulate in liver and tumor. The correlation coefficient of tumor volumes done by three-dimensional ultrasound and at autopsy was 0.997. In efficacy, tumor volume in the normal saline-treated group was 1803.2 mm3 at 54 days after tumor inoculation. Tumor volumes in the 103.6 MBq and 240.5 MBq of 188Re-HSAM treated groups were 381 and 267.4 mm3 (p = 0.001 and 0.004), respectively. CONCLUSIONS: These results show that three-dimensional ultrasound with a high spatial resolution and contrast in soft tissue can become imaging modality in assessing tumor burden and tumor progression in an orthotopic rat model. The longitudinally therapeutic evaluation of 188Re-HSAM demonstrated dose-dependent tumor growth inhibition with increased dose in the GP7TB orthotopic hepatoma rat model.

Sequence diversity and associated pathogenicity of the hemagglutinin cleavage site of H5N2 avian influenza viruses isolated from chickens in Taiwan during 2013-2015.

The sequence at the hemagglutinin (HA) cleavage site (CS) plays a key role in determining the pathogenicity of avian influenza viruses. Three types of HA CS sequences, QREKR/GL, QRKKR/GL and QRRKR/GL, were previously reported in Taiwanese H5N2 viruses that were isolated from chickens from 2003 to 2013. However, no HA CS sequence was reported for viruses isolated after 2013. This article presents the HA CS sequences and pathogenicity of H5N2 viruses that were isolated from chickens in Taiwan during 2013-2015. Two novel HA CS sequences, QKEKR/GL and KREKREKR/GL, were found in the viruses isolated in 2013 and 2014, and pathogenicity tests showed that the viruses with these novel HA CS sequences are low and high pathogenic viruses, respectively. In contrast, the HA CS sequence QREKR/GL was found in all viruses that were isolated in 2015, and all of these viruses were low pathogenic viruses. After 10 passages in embryonated chicken eggs, a virus strain that was isolated in 2003 evolved into a viral quasispecies that contained at least four distinct types of HA CS sequences. These results highlight the potential of Taiwanese H5N2 viruses to change their pathogenicity and HA CS sequences via mutations. Furthermore, viruses with the HA CS sequence QREKR/GL were more prevalent than others in 2015. These findings are useful for understanding the mechanism of sequence changes at the HA CS and for refining H5N2 virus control measures in Taiwan.

Differential expression of U2AF35 in the arthritic joint of avian reovirus-infected chicks.

To identify cell types and genes that are differentially expressed during immunopathogenesis of avian reovirus (ARV)-induced viral arthritis (VA), we inoculated arthrotropic strain S1133 of ARV into 1-day-old broilers, and examined tissue histology as well as RNA expression at different days post-inoculation (PI). Using immunohistochemical staining, we detected many CD68 expressing macrophages in and around the blood vessels of the arthritic joints. By RT-PCR, we found that expression of matrix metalloproteinase-2 (MMP-2) and bone morphogenetic protein-2 (BMP-2) was induced earlier in footpads and hock joints of ARV-infected chickens. By employing suppression subtractive hybridization (SSH) technique and RT-PCR, we further identified that small subunit of U2 snRNP auxiliary factor (U2AF35 or U2AF1) mRNA was differentially induced in the joint of ARV-infected chickens. By in situ hybridization (ISH), mRNA signals of U2AF35 and BMP-2 were located in chondrocytes within/near the epiphyseal plate and secondary center of ossification, and in epidermal cells and dermal fibroblast-like cells of arthritic joints. In addition, U2AF35 mRNA was expressed in the inflammatory infiltrates of the bone marrow of ARV-infected arthritic joints, while MMP-2 was mainly detected in chondrocytes. Interestingly, among U2AF35, MMP-2, and BMP-2 that were differentially expressed in the joint of ARV-infected chickens, only U2AF35 induction correlated well with arthritic manifestation. Because U2AF35 may assist in mRNA splicing of proinflammatory chemokines and cytokines, our results indicated that U2AF35 induction might play an immunopathological role in ARV-induced arthritis. This study has first associated U2AF35 to viral arthritis.

Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005.

This article reports the genetic and pathogenic characteristics of 34 isolates of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Genetic analyses showed that a unique lineage of H6N1 viruses has been established in domestic chickens in Taiwan since 1997, and this lineage of viruses differs from the H6N1 viruses circulating in Hong Kong and Southeastern China. Pathogenicity tests showed that all Taiwanese H6N1 viruses were of low pathogenicity but might lead to economic loss when associated with other diseases. Hemagglutination inhibition tests showed that antigenic drift has occurred in Taiwanese H6N1 viruses, and sequence comparison has identified a total of five possible antigenic sites on the hemagglutinin molecule of the H6N1 viruses. Some Taiwanese H6N 1 viruses could replicate in mice without preadaptation, indicating that these viruses have the potential to cause cross-species infection into mammals.

Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus.

This article reports the complete nucleotide sequences of four duck circovirus (DuCV) isolates from sick ducks in Taiwan and development of a polymerase chain reaction (PCR) for detection and differentiation of goose circovirus (GoCV) and DuCV. Sequence comparison showed that Taiwanese DuCV isolates had 82.5%-83.8% nucleotide sequence identity to the German and North American DuCV isolates. This is the first report on the presence of DuCV and its associated diseases outside Germany. A PCR test was developed using a universal primer pair based on conserved sequences present in the genomes of GoCV and DuCV. This PCR test could detect and differentiate between GoCV and DuCV by the size of PCR product each virus produced (256 bp for GoCV and 228 bp for DuCV). Application of this PCR test to samples of bursa of Fabricius from sick birds in the field showed that 9 of 26 goose samples contained GoCV, while 13 of 34 duck samples contained DuCV. This PCR test could serve as a fast and sensitive method for detection and differentiation of DuCV and GoCV.

Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida.

Synergistic effects between the same class of antibiotics are rarely reported. Our previous study found synergistic-like interaction between florfenicol (FFC) and thiamphenicol (TAP) against Staphylococcus aureus. Here, the enhanced antimicrobial activity was evaluated in 97 clinical isolates of both Gram-negative and Gram-positive bacteria. Susceptible strains were initially identified by checkerboard microdilution assay (fractional inhibitory concentration index [FICI] ≤ 0.625), followed by confirmation of synergism using the time-kill methodology (≥2 log10 CFU/ml reduction). In all, 43% of Pasteurella multocida tested were susceptible to the enhanced bactericidal effect. In chicken fowl cholera models, FFC and TAP combination at much lower dosage that is correspondent to their MIC deduction provided maximum protection in vivo. Furthermore, synergistic combination of FFC with oxytetracycline (OTC) against Pseudomonas aeruginosa in vitro was also demonstrated. Based on the enhanced uptake of TAP and OTC, FFC presumably elicits enhanced antimicrobial activity in an orderly manner through alteration of bacterial membrane permeability or efflux systems and subsequent increase of intracellular concentration of the antibiotics used in combination. Results of ethidium bromide accumulation assay and RNA-seq showed little evidence for the involvement of efflux pumps in the synergy but further investigation is required. This study suggests the potentiality of a novel combination regimen involving FFC as an initiating modulator effective against both Gram-positive and Gram-negative bacteria depending on the antibiotics that are combined. The observed improvement of bacteriostatic effect to bactericidal, and the extended effectiveness against FFC-resistant bacterial strains warrant further studies.

Characterization of functionally active interleukin-18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells.

The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL-18a cell line that stably expresses the fusion protein chIL-18 was constructed and the enhanced green fluorescence protein connected through a (G4 S)3 linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL-18a cells (1 × 10(5) ) were inoculated in 60-mm culture dishes containing 5 mL of media to achieve 50%-60% confluence and were cultured in the presence of the cycle-specific inhibitors 10058-F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058-F4, aphidicolin, and colchicine, respectively, the aphidicolin-induced single cells showed higher fusion protein levels than did the 10058-F4- or colchicine-induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin-treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN-γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581-591, 2016.

Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12.