Effect of turning upon the sub‐embryonic fluid and albumen of the egg of the Japanese quail

1. The effects of egg turning upon the formation of sub‐embryonic fluid (SEF), and albumen weight and composition are described up to day 8 of incubation.

2. The density of albumen increased, and the density of yolk sac contents decreased, during the first 5 d of incubation. Failure to turn eggs diminished these changes.

3. The rate of formation of SEF and its maximum weight were lower if eggs were not turned, as was the rate of decrease in albumen weight.

4. The concentration of sodium and chloride, as well as osmolality, were higher in SEF than in thin albumen, and were affected by a failure to turn eggs.

5. Static incubation altered the concentrations of yolk nutrients in SEF.

6. It is concluded that the formation of SEF is primarily dependent upon the transfer of sodium and chloride from albumen to SEF so creating an osmotic force for water movement in the same direction. Turning the eggs promotes this process by ensuring an adequate supply of ions in thin albumen adjacent to the blastoderm.     

Molecular identification of Cryptosporidium spp. from fecal samples of felines, canines and bovines in the state of São Paulo, Brazil

The objective of this study was to obtain information of epidemiological nature through genotypic characterization of Cryptosporidium isolates from dogs, cats and bovines from the state of São Paulo, Brazil. The extraction of DNA from oocysts was carried out and polymerase chain reaction was accomplished using specific primers to 18S rRNA gene. The amplicons were directed sequenced. Seven cat samples, nine dog samples and nine bovine samples were analysed. From the seven cat samples the genotypic analyses revealed Cryptosporidium felis in all. These were the first genotypic characterization of Cryptosporidium from domestic felines in Brazil. In nine sequenced samples from dogs, genotypic identities compatible with Cryptosporidium canis were revealed in all samples. The genotypic analyses in bovines revealed Cryptosporidium parvum in eight samples and Cryptosporidium bovis in another sample, the last one being a non-zoonotic species, not related to clinical symptoms and described for the first time in Brazil.     

Molecular cloning of the swine interleukin-23 subunit p19 and of its receptor components interleukin-23Rα and -12Rβ1

Interleukin (IL)-12 and IL-23 play central roles in the regulation of distinct helper T-cell subsets, i.e. Th1 and Th17, respectively. Although IL-12 and IL-23 have been well studied in human and rodent systems, little is known about their significance in other animals, including livestock mammals such as cattle and pigs. In this study, we performed molecular cloning and genetic characterization of a small component of swine IL-23, i.e., IL-23p19; in addition, we identified and performed chromosomal assignment of the genes encoding its receptor (R) subunits IL-23Rα and IL-12Rβ1. These results provide genetic information about both swine IL-23/IL-23R and IL-12/IL-12R systems, which allows for better understanding of IL-12/IL-23 systems involved in pig immunity.     

Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25)

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.     

GeneScan analysis to detect clonality of T-cell receptor γ gene rearrangement in feline lymphoid neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. Polymerase chain reaction (PCR) amplification of the complementarity-determining region (CDR) 3 of the T-cell receptor (TCR) γ gene can be used to assess clonality of T-cell populations as a supportive diagnostic tool for T-cell neoplasms. Because the length variation in the TCRγ CDR3 is relatively small, false positive results may occur in non-neoplastic T-cell populations in the absence of high-resolution analytical methods for PCR products. In the present study, a PCR assay system was developed to detect clonal TCRγ gene rearrangement in feline lymphoid cells using GeneScan analysis. Thirty T-cell neoplasms, 27 B-cell neoplasms, and 34 non-neoplastic tissues were subjected to the newly developed TCRγ gene rearrangement analysis. Clonal TCRγ gene rearrangement was detected in 26 of 30 (87%) T-cell neoplasms, 2 of 27 (7%) B-cell neoplasms, and 1 of 34 (3%) non-neoplastic tissues. To compare GeneScan analysis with conventional PAGE and heteroduplex analysis, 20 clonal and 20 polyclonal samples were subjected to both analyses. Most of the results were concordant between the 2 analyses; however, several clonal peaks (bands) appeared as a single band when analyzed via conventional PAGE with heteroduplex analysis in 4 of the 20 (20%) clonal samples as a result of the difference in resolution. The PCR assay system to detect clonal TCRγ gene rearrangement in feline lymphoid cells, using GeneScan analysis, would be a useful molecular diagnostic tool for feline T-cell neoplasms, with high fidelity.     

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a 'memory' phenotype, being CD62L(-)/CD45RA(-) and expressing high levels of CD2 and CD11a; efferent cells are largely 'na?ve', being CD62L(+)/CD45RA(+) with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNgamma, TGFbeta and TNFalpha) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments.     

Molecular cloning, in vitro expression and bioactivity of rabbit transforming growth factor-beta receptor type II (rTGF-βRII)

Transforming growth factor-beta receptor II (TGF-βRII) is an attractive target for anti-scarring therapy in wound healing because it attenuates excessive TGF-β which has pleiotropic effects on the immune system. In the present study, the cDNA of rabbit TGF-βRII (rTGF-βRII) was amplified from rabbit peripheral blood by RT-PCR. The open reading frame of rTGF-βRII encodes a protein consisting of 567 amino acids, which contains a predicted transmembrane domain and a Serine/Threonine protein kinase domain similar to other identified mammalian TGF-βRIIs. The amino acid sequences of the biologically active, soluble rTGF-βRII and mouse, rat, human and chicken counterparts are 81%, 81%, 89% and 61%, respectively, identical. Recombinant soluble rTGF-βRII (rsTGF-βRII) fused with His tag was efficiently expressed in Escherichia coli BL21 (DE3) expression host strain. This fusion protein's molecular weight of ～ 19 kDa was identified by SDS-PAGE and Western blotting. In vitro, purified rsTGF-βRII was able to inhibit the proliferation of keloid rabbit fibroblasts and decrease the level of collagen. These findings indicate that rTGF-βRII plays an important role in inhibiting the proliferation of keloid rabbit fibroblasts and provides the basis for investigations on the role of TGF-βRII in this important domestic species.     

Molecular identification of polydorid polychaetes (Annelida: Spionidae): is there a quick way to identify pest and alien species?

The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among species. This taxon will therefore benefit from the development of a library of sequences, such as COI barcodes, to aid identification. However, the universal primers for the cytochrome c oxidase I (COI) DNA barcoding marker has failed to consistently amplify this gene for polydorids, greatly hampering the development of such a library. We describe the development of unique PCR primers for the COI gene that work across four genera and nine species of polydorids. We also compared its efficacy with sequence data for mitochondrial cytochrome b and nuclear 18S rRNA, and a concatenated dataset consisting of all three markers. The nuclear 18S rRNA gene showed the least variation both intra- (0.0–1.2%) and interspecifically (0.6–4.3%), and was the most accurate for species identifications among the three markers. Although COI was characterised by higher intraspecific variation compared with Cyt b (0.0–14.5% and 0.0–4.2%, respectively), Cyt b showed considerably higher levels of interspecific variation (16.6–30.2%) compared with COI (2.2–20.7%). Of the two mitochondrial DNA markers, COI was actually less accurate for species identifications, having suggested two species within Boccardia pseudonatrix that was not supported by the other markers. Overall, the concatenated dataset yielded the most consistent intraspecific groupings, suggesting that this is the most accurate means of identifying polydorids using DNA sequence data. Thus, there may not be a quick and easy way to identify these species accurately using only molecular data.     

Promoter identification and effect on activation of NF-κB of porcine ISG58

Interferon (IFN)-stimulated gene (ISG) 56 family (composed of ISG54, ISG56, ISG58, and ISG60) plays important roles in defense against viral infection in mammalian cells. Numerous studies have been conducted on ISG54, ISG56, and ISG60; however, little is known on ISG58. In the present study, the upstream sequence of porcine ISG58 gene was first characterized as functional promoter by luciferase reporter assay, and then two directly adjacent IFN-stimulated response elements (ISREs), one at ?206 to ?194 (ISRE-I) and a second one, directly upstream of this element at ?219 to ?207?bp (ISRE-II), were identified using the bioinformatics method. The subsequent site-directed deletion and transient transfection experiments showed the candidate ISREs are functional. ISRE-I works better than ISRE-II and synergistic cooperation exists between two ISREs. Additionally, the effect of porcine ISG58 on activation of NF-κB was analyzed using the dual-luciferase reporter assay. The results will contribute to revealing the role of ISG58 in immune response.     

Influence of in ovo injection of disaccharides, glutamine and β-hydroxy-β-methylbutyrate on the development of small intestine in duck embryos and neonates

1. The objective of the present study was to examine the effect of in ovo injection of disaccharides (DS), disaccharides and glutamine (DS + Gln) or disaccharides and β-hydroxy-β-methylbutyrate (DS + HMB) at d 23 of incubation on the development of the small intestine. 2. In DS + Gln-injected ducks, the greatest relative small intestine mass and muscularis layer thickness among 4 treatments was observed from d 25 of incubation to 7 d of age. 3. Jejunal sucrase activity in DS-injected ducks was significantly greater than in controls at hatch and on d 7. 4. In DS + HMB-treated ducks, a tendency toward slightly higher jejunal DNA concentration was observed throughout the experiment. 5. Greater body weight was found in DS + Gln and DS + HMB treated ducks in the first two weeks. However, there was no significant difference in the market weight (35 d) of ducks among the 4 treatments. 6. The results of present study suggest that administering disaccharides and Gln, or disaccharides and HMB, to the duck embryos exerted a beneficial effect on the early development of small intestine and on growth performance.     

Molecular weight and amino acid composition of fowl delta‐globulin

1. The molecular weight of delta‐globulin was estimated chromato‐graphically to be about 10,700 daltons.

2. Ultracentrifuge experiments at 2.7 × 10⁵ g gave values of s _{20, W} = (1.49 + 0.16C) × 10^?13 s for the sedimentation coefficient and D_{20 w} = (1.12 + 0.19c) × 10^?6 cm² s^?1 for the diffusion coefficient, c being the protein concentration (g/100 ml).

3. According to the Svedberg equation, these imply a molecular weight of 12,470 daltons, assuming [v_bar] = 0.74 ml/g.

4. Using refractometer measurements of protein concentration it was found that E ^1% _1cm (278 nm) = 5.57 at pH 7.8.

5. From the ultraviolet absorption spectrum of the protein in 0.1 N NaOH it was concluded that the molecule probably contains four tyrosine residues and no tryptophan.

6. On this basis a revised amino acid composition is given.     

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine^®, 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.