Chick Kidney Cells Research Articles

Three-fold induction of renal 25-hydroxyvitamin D3-24-hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture.

To investigate the possible biological actions of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), a tibial fracture-healing model was established in White Leghorn chicks. Three-week-old White Leghorn chicks fed a vitamin D3-replete diet were divided into four groups (control, anesthetized, sham, and fractured). On varying days after tibial fracture (F) or sham manipulation (S), renal 25(OH)D3-1 alpha-hydroxylase and 25(OH)D3-24-hydroxylase (24-hydroxylase) activities and serum Ca2+ concentrations were measured. Metofane anesthesia was found to have no effect on the activity of either of the hydroxylases; the activities of the hydroxylases in the control, anesthetized, and sham-operated birds were similar. By 10 days after tibial fracture, the renal 24-hydroxylase activity increased more than 3-fold in F (1.33 +/- 0.07 pmol/mg of protein) as compared with S (0.42 +/- 0.03 pmol/mg of protein) (p < 0.0001). A time-dependent study of the renal 24-hydroxylase activity during the fracture repair process revealed a slow increase from the first day after fracture, a higher activity at 8 days, which peaked at 10-11 days, which is consistent with the formation of the callus. The 24-hydroxylase activity then returned to the same level as the sham group 14 days after fracture. There was no significant difference in serum Ca2+ levels between the F and S groups over the 3-week postfracture period. Serum levels of vitamin D3 metabolites were also measured during the fracture healing process: a 3.4x increase of the 24,25(OH)2D3 level in the fractured group (3.64 +/- 1.16 nM) was observed as compared with the control groups (1.08 +/- 0.49 nM) at 10 days after fracture (p = 0.068). No significant differences were observed in the plasma levels of 25(OH)D3 or 1 alpha, 25(OH)2D3 between the group with a fracture and the controls. Exposure of primary chick kidney cells in culture to serum obtained from chicks with a tibial fracture for 20 h resulted in an approximately 40% increase in the activity of the 24-hydroxylase as compared with cells exposed to serum from control birds. These results suggest that 24,25(OH)2D3 is involved in the early process of fracture repair and that there is some form of physiological communication between the fractured bone and the kidney so as to increase the renal 24-hydroxylase and the circulating concentration of this metabolite.

Adapting Eimeria tenella to Grow in Primary Chicken Kidney Cells Following Repeated Passages between Cell Culture and Chickens

The present study was undertaken to adapt a field isolate (F0) of Eimeria tenella to grow in primary chicken kidney cells (PCKCs) by selecting for characteristics of the parasite rather than modifying the culture and/or environmental conditions. Fourteen generations (F1 to F14) of E. tenella were produced following repeated passages between PCKCs and chickens. Although F1 yielded only a 28% increase in oocysts, in PCKCs compared with F0, F2 to F5 produced from 259% to 277% more oocysts, respectively. There was no significant increase in the percentage of oocysts produced in PCKCs by F6 to F14 compared with F5. Generations F1 to F14 demonstrated a greater propensity for multiple infections within the same host cell than did F0. For example, it was not uncommon to observe two, three, and occasionally four oocysts within a single PCKC. Chickens inoculated with F0 oocysts generally experienced greater pathogenesis by day 7 postinoculation than chickens inoculated with F14 oocysts as measured by decreased body weights, increased cecal lesions, and a higher mortality rate.

Monoclonal antibodies inhibiting invasion of cultured cells by Eimeria tenella sporozoites.

Monoclonal antibodies (MoAbs) secreted by 12 hybridomas and reactive with the surface antigens of E. tenella sporozoites were produced. These MoAbs designated as KC-1 to KC-12 were characterized as IgG3 kappa. In Western blot analysis, these MoAbs reacted with only one polypeptide band of sporozoite antigens having a molecular mass of 25 kDa. All the MoAbs were reactive with sporozoites, trophozoites, immature and mature first generation schizonts, first generation merozoites, and the interior structure of sporulating oocysts of E. tenella, however, not with any of the methanol-fixed sporozoites of E. acervulina, E. mitis, E. maxima, E. brunetti, E. necatrix and E. praecox. Invasion of primary culture of chicken kidney cells by E. tenella sporozoites was significantly inhibited by several of the MoAbs. These results suggest that these MoAbs recognizing the E. tenella-specific surface molecule are involved in the invasion of sporozoites into host cells.

Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.

The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

A single gene encoding the fiber is responsible for variations in virulence in the fowl adenoviruses.

Intertypic recombinant fowl adenoviruses (FAVs) were generated to determine regions of the viral genome involved in virulence. Recombinants were produced with two serotype 8 FAVs, mildly virulent CFA 3 and hypervirulent CFA 40. Restriction endonuclease fragments from the genomes of the two FAVs were used to transfect primary chicken kidney cells. Virulence testing of these recombinants located the region responsible for differences in virulence to an 8.4-kb fragment of the genome located between kb 26.6 and 35.0. According to data available for a serotype 10 FAV that had been partially characterized in the laboratory, this segment of the genome contained three genes of known identity (100K, 33K, and pVIII) and a region between kb 31 and 35 with unknown coding potential (although this information subsequently became available for a serotype 1 FAV, CELO). Therefore, the region between kb 30.5 and 34.5 was sequenced. The results revealed that the unknown region encoded a fiber gene on the right strand and several small open reading frames of unknown identity on the left strand. Further recombinant viruses containing defined exchanges within the 4-kb fragment were constructed, and virulence testing of these viruses indicated that the fiber was responsible for differences in virulence for CFA 40 and CFA 3.

Sequence comparisons of A/AA/6/60 influenza viruses: mutations which may contribute to attenuation

Influenza virus infection is a worldwide public health threat. Cold-adaptation was used to develop a vaccine line (ca A/AA/6/60 H2N2) which promised to reduce the morbidity and mortality associated with influenza and to serve as a model for other live virus vaccines. This study establishes that two distinct lines of wt A/AA/6/60 viruses exist with different phenotypic and genotypic characteristics. The two virus lines have the same parent but different passage histories. The first line is both temperature sensitive (ts) and attenuated in ferrets and the second line (after multiple passages in chick kidney cells, eggs and mice) is non-ts and virulent in ferrets. Both lines of viruses have been further differentiated by sequence analysis. We have identified point mutations common to all virulent viruses but absent from the attenuated viruses. This was accomplished by comparing the nucleotide sequences of the six internal genes in three different attenuated passages of A/AA/6/60 with those of five different virulent passages of the same virus. The corresponding nucleotides of the attenuated viruses, therefore, represent candidate attenuating lesions: 6 in the basic polymerase genes (5 in PB1, 1 in PB2), 2 in the acidic polymerase gene (PA), 1 in the matrix (M) gene, 2 in the non-structural (NS) gene, and none in the nucleoprotein (NP) gene. Two of the 5 attenuating lesions in PB1 are silent; 1 2 in PA is silent; and 1 2 in NS is silent. Further changes which might be identified by comparing nucleotide and amino acid sequences of the A/AA/6/60 viruses with those of other influenza viruses may also contribute to the attenuation of the ca virus. Our study identifies nucleotides which more precisely define virulence for this virus and suggests that growth of the virus at low temperature may have preserved a non-virulent virus population rather than attenuating a virulent one.

Eimeria tenella:Role of Carbohydrates on Sporozoite at the Penetration into Cultured Cells

The effects of carbohydrates on the penetration ofEimeria tenellasporozoites into cultured cells were investigated. Eight different carbohydrates that have been known to be on the surfaces of animal cells were added to culture medium during the penetration period.d-Galactose alone inhibited sporozoite penetration into primary chicken kidney cells. When kidney cells were pretreated withd-galactose and infected withE. tenellasporozoites, the penetration was suppressed significantly, but not when the sporozoites were pretreated. In addition, the penetration of sporozoites was suppressed when pretreated with peanut lectin that specifically recognizesd-galactose residues at a concentration of 50 μg/ml. The present results suggested thatd-galactose residues onE. tenellasporozoite surfaces and the lectin-like receptors that recognized-galactose of host cells are important factors for penetration.

Immunochemical study of a transforming growth factor-α-related protein in the chicken kidney

A number of polypeptides are involved in renal growth and physiology. Both transforming growth factor-alpha (TGF-alpha) protein and mRNA are expressed in kidney cells during embryonic and adult stages, and exert mitogenic activity on kidney cells in culture. We studied the immunolocalization of a TGF-alpha-related protein at the ultra-structural level and found it in the basolateral membranes of dark cells from distal tubules of the chicken kidney. By Western blotting techniques, we identified a protein complex composed of a least two TGF-alpha immunoreactive subunits of 40 and 88 kDa, respectively. Both subunits were sensitive to elastase digestion, and released TGF-alpha immunoreactive products. In addition, TGF-alpha immunoreaction was found in primary culture of chicken kidney cells. These findings suggest that the TGF-alpha-related protein complex plays a very specific role in proliferation and/or differentiation of kidney cells.

Antagonistic effect of nucleic acid precursors on the anticoccidial activity of 6-azauracil.

The antagonistic phenomena to anticoccidial activities of 6-azauracil (AzU) induced by certain nucleic acid precursors were examined in battery experiments. Each nucleic acid precursor, orotate, orotidine, uracil, uridine, adenine and adenosine was mixed in feed in combination with effective levels of AzU. The test feed was provided through the test term to White Leghorn cockerels which were infected with Eimeria tenella, E. necatrix or E. acervulina. In vitro tests were also conducted for antagonistic phenomena similarly to in vivo test using E. tenella cultured in chick kidney (CK) cells. Orotate and uridine reduced dose-dependently the anticoccidial activity of AzU in E. tenella infection in chickens. Uracil and orotidine revealed similar effect. Adenine and adenosine did not show any antagonistic effect to AzU. In E. necatrix infection, orotate and uridine had a similar tendency to that of E. tenella, whereas uracil and orotidine was less pronounced. The activity of AzU against E. acervulina was also blocked by the treatment with uridine and orotate. AzU inhibited the schizont's development of E. tenella in the CK cells, and this effect was reduced by uridine, but not by orotate and orotidine. The antagonistic effect of the pyrimidine nucleic acid precursors on AzU activity suggested that the anticoccidial effect of AzU to E. tenella, E. necatrix and E. acervulina is derived from inhibition of the metabolism of pyrimidine nucleotide synthesis in these parasites.

Characteristics of the anticoccidial activity of 6-azauracil.

The characteristics of anticoccidial activities of 6-azauracil (AzU) were investigated in the battery trials utilizing the White Leghorn cockerels, which were infected with Eimeria tenella, E. necatrix, E. acervulina, E. maxima and/or E. brunetti. AzU was mixed into the basal starter feed and fed ad libitum to the birds from 1 day before the inoculation of oocysts to the time of necropsy. AzU showed remarkable anticoccidial activity against E. tenella and E. necatrix infections at doses of 1,000 ppm in feed or more, and fairly good effect against E. acervulina infection at the dose of 4,000 ppm in feed. Inadequate effect against E. maxima infection and null in effect against E. brunetti at the dose of 4,000 ppm were resulted for this drug. The effect of 6-azauridine, ribonucleoside of AzU against E. tenella and E. necatrix infections at 1,000 ppm was tested and revealed negative results. In the test with AzU-resistant line of E. tenella, cross resistance was confirmed between AzU and emimycin riboside, a uridine analogue. Activity of AzU was studied in vitro against E. tenella in chick kidney cells. AzU inhibited the development of the first- and second-generation schizonts at 100-200 ppm in the medium. Degenerated parasites were observed.

Increasing the Yield of Eimeria tenella Oocysts in Primary Chicken Kidney Cells

The present study was undertaken to increase the yield of Eimeria tenella oocysts in primary chicken kidney cells (PCKCs) using a comparatively inexpensive cell-culture system. PCKCs growing on coverslips positioned on the bottoms of individual wells in 24-well tissue-culture plates were infected with sporozoites of E. tenella. The effects of changing the culture medium (RPMI 1640), medium pH, serum type, and serum concentration in the wells were determined by counting newly produced oocysts at 7 days postinoculation. There were significantly more (P < 0.01) oocysts produced when the medium was supplemented with 10% fetal bovine serum (FBS) and changed either daily or every other day compared with not changing the medium. When the same medium was changed daily, significantly more (P < 0.05) oocysts were produced at pH 7.4 than at pH 8.0 but not at pH 6.0. If the medium was changed daily, significantly more (P < 0.05) oocysts were produced when medium was supplemented with 10% FBS only rather than 5% and 10% chicken serum. The cell-culture system described in this study offers a convenient and efficient method for investigating the biological, biochemical, and immunological relationships between E. tenella and the host cell.

A Highly Sensitive, Broad-Spectrum Infectivity Assay for Infectious Bursal Disease Virus

In order to develop an infectivity assay for infectious bursal disease virus (IBDV), an enzyme-linked immunosorbent assay (ELISA) for detecting IBDV antigens was developed using a rabbit antiserum to IBDV. The ELISA detected both serotypes 1 and 2 of IBDV, purified virus proteins at a concentration of about 0.1 ng/well and about 10(4) to 10(5) infectious units/well, and was about 10(3) times more sensitive than an agar gel precipitin test. By using the ELISA for detecting IBDV antigens in the cultured fluids, infectivity titration could be determined within 5 days of cultivation in seven of the 10 virus-cell combinations when chicken embryo fibroblasts (CEFs), BGM-70 cells, and LSCC-BK3 cells were used. IBDV antigens were not detected in three combinations remaining after 7 days of cultivation. When 18 IBDV strains, including highly virulent, classically virulent, and attenuated strains, were tested for growth in four types of cells using the ELISA, eight strains in CEFs, seven strains in chicken kidney cells, five strains in BGM-70 cells, and 17 strains in LSCC-BK3 cells were detected. These results indicated that LSCC-BK3 cells had the broadest spectrum for IBDV. When 26 field bursal homogenates were tested for infectious IBDV using LSCC-BK3 cells, all 19 field IBDV isolates that were detected by immunostaining of the cells were also detected by the ELISA. These results indicate that this infectivity assay for IBDV using LSCC-BK3 cells and the ELISA has a high sensitivity and a broad spectrum for IBDV and is especially useful for large-scale applications.

Generation of a defective RNA of avian coronavirus infectious bronchitis virus (IBV). Defective RNA of coronavirus IBV.

The Beaudette strain of IBV was passaged 16 times in chick kidney (CK) cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage six. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12423 to 18744 in the IBV genome and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Confocal Laser Scanning Microscopy of β-Tubulin and α-Actinin in Asexual Stages of Eimeria tenella (Apicomplexa: Eimeriidae) in Cell Culture

Summary: Specific monoclonal antibodies were used to visualize the dynamics of β-tubulin and α-actinin in asexual development of Eimeria tenella in cultured primary chicken kidney cells by means of confocal laser scanning microscopy. β-tubulin was more condensed in refractile bodies (RB), at anterior ends, and along subpellicles in sporozoites. The RB remained labelled during the development of first generation schizonts until the formation of merozoites, suggesting a possible important role of RB in the reorganization of microtubules in schizonts. The microtubules also formed a number of tiny aggregation centers scattered throughout the schizonts corresponding to the formation of first generation merozoites. The microtubular material in second generation schizonts was clustered around several weakly-stained areas. Both first and second generation merozoites had more highly concentrated β-tubulin at anterior ends and along subpellicles. In contrast, α-actinin, and actin-binding protein, was more concentrated in the anterior half area of sporozoites. It became evenly distribution in the whole cytosol of developing trophozoites except RB areas. The distribution of α-actinin in first or second generation schizonts corresponded to merozoite formation. The β-tubulin and α-actinin of chicken primary kidney cells were also labelled by the two monoclonal antibodies.

Oscillatory behavior of control-systems of calcium homeostasis in chickens.

Computer simulation of calcium homeostasis in chicks predicted an oscillatory behavior of bone calcium flow and kidney 25-hydroxyvitamin D3-1-hydroxylase with a periodicity of 56 h and a 9 h phase difference between the two signals. In growing chickens subjected to a light: dark cycle of 22:2 h, and intravenously dosed with 45Ca, the temporal changes in plasma 45Ca could be described by an exponential decline with superimposed diurnal oscillations. The activity of the renal 25-hydroxyvitamin D3-1-hydroxylase in chicks subjected to a 12:12 h light: dark cycle ALSO followed diurnal oscillations, with a nadir at the beginning of the light period and a peak 12 h later. The production of 1,25-dihydroxyvitamin D3 by primary cultures of chicken kidney cells oscillated with a periodicity of 5.6 h or shorter. It is suggested that despite the differences in phase and periodicity between the simulation predictions and actual results, the oscillations in both 1-hydroxylase and bone calcium flow could be coupled through the hormonal systems involved in regulation of plasma calcium.

Characterization of a Replicating and Packaged Defective RNA of Avian Coronavirus Infectious Bronchitis Virus

The Beaudette strain of IBV was passaged 16 times in chick kidney cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32 P-labeled cDNA probes corresponding to the first 2 kb of the 5′ end of the genome, but excluding the leader, and to the last 1.8 kb of the 3′ end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage 6. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5′ end of the genome, 6322 from gene 1b corresponding to position 12,423 to 18,744 in the IBV genome, and 1626 from the 3′ end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Inhibition of the in vitro Development of Eimeria tenella in Chick Kidney Cells by Immune Chicken Splenocytes

An in vitro microbicidal assay was used to study the immune response of chickens to Eimeria tenella by measuring the effect of splenocytes from immunized chickens on intracellular development of E. tenella. Splenocytes were prepared from specific-pathogen-free chickens [strain P2a(B19B19) or N2a(B21B21)], immunized one, two, or three times with non-lethal doses of E. tenella. Twelve hours following infection of chick kidney cells (CKCs) with E. tenella sporozoites, splenocytes were added to infected CKCs for 4, 8, or 36 hours. Intracellular E. tenella development was allowed to continue until 72 hours after sporozoite infection, when intracellular development was quantitated by counting merzoites. Immune splenocytes significantly inhibited E. tenella intracellular development after one, two, or three immunizations. Significant inhibition occurred with 4, 8, or 36 hours of coculture and was no greater with longer co-culture times. Immune P2a splenocytes significantly reduced merozoite development in both syngeneic P2a and allogeneic N2a infected CKCs, whereas immune N2a splenocytes had little effect on E. tenella development in either N2a or P2a infected CKCs. These results suggest that immune splenocytes are induced and act relatively rapidly and are not apparently restricted by the major histocompatibility complex, consistent with natural killer cell activity.

Left ventricular endocardial proliferation in chickens.

Left ventricular endocardial proliferation in chickens.

Peptides Associated with Monensin Resistance in Sporozoites of Eimeria tenella (Coccidia)

Resistance to monensin in the sporozoites of a laboratory strain (WIS) of Eimeria tenella was amplified by treating free sporozoites with increased monensin levels in vitro, followed by propagation of these treated sporozoites in chickens. The parent strain of WIS and its subsequent lines developed from the treatment of monensin at 1, 5, or 25 micrograms/ml were designated as WIS(0), WIS(1), WIS(5), or WIS(25), respectively. The penetration rate of sporozoites into primary chicken kidney cell cultures showed that the sensitivity of sporozoites to the treatment of monensin at 1 and 5 micrograms/ml was significantly reduced in the WIS(25) line in comparison with the WIS(0) line. When native polyacrylamide gel electrophoresis (PAGE) was conducted, a change in the relative mobility of a protein band was found in the protein samples of these coccidial lines. Sodium dodecyl sulfate PAGE revealed that 2 peptides with molecular weights of approximately 50.0 and 31.4 kDa were present in the sporozoites of resistant lines but undetectable in their WIS-parent sporozoites. Derivation of the resistant lines from a drug-sensitive parent line gave strong support to a link between the appearance of the peptides and resistance to ionophores in this strain of E. tenella.

Characterization of Marek's Disease Virus BamHI-A-Specific cDNA Clones Obtained from a Marek's Disease Lymphoblastoid Cell Line

A cDNA library was constructed from poly(A) + RNA fractions obtained from a Marek's disease (MD) lymphoblastoid cell line, MDCC-CU41, in which viral gene expression is very limited. Three independent groups (1, 2, and 3) of MD virus (MDV)-specific clones were obtained, which were mapped in the inverted repeat region of the BamHI-A fragment of the MDV genome. Northern blot analysis showed that probes prepared from these cDNA clones hybridized with several transcripts of different sizes in poly(A) + RNA of MDCC-CU41, although the amounts of these transcripts were relatively small compared to those in MDV lytically infected cells. Moreover, a small open reading frame, which can encode a 94-amino-acid protein, was identified in the A41 cDNA clone (Group 3). By RNase protection assays, the 1.2-kb Group 3 transcriptional unit has been defined. In indirect immunofluorescent antibody assays, antiserum against the bacterially expressed fusion protein, glutathione S -transferase-A41, reacted specifically with the cytoplasmic regions of MDV (strain RB1B)-infected chick kidney cells. However, MDCC-CU41 did not contain a detectable lever of the protein determined by these methods.