Characterization of cecal microbiota and response to an orally administered lactobacillus probiotic strain in the broiler chicken

A probiotic Lactobacillus strain was given in drinking water to young broiler chickens from 1 to 19 days of age. Cecal contents were collected from 4- and 19-day-old chickens in treated and control groups. Enumeration of bacteria by culture on selective media showed a decrease in Clostridium perfringens carriage in the 4-day-old treated chickens, whereas coliforms and Lactobacillus populations were not significantly affected by the treatment. Fluorescent in situ hybridization analysis with 7 phylogenetic probes targeting the major groups of intestinal bacteria revealed that the Clostridium coccoides group accounted for more than 50% of the total bacteria in the cecum of 4-day-old chickens, whereas the bacterial community of 19-day-old chickens evolved towards a more diverse microbiota with Faecalibacterium prausnitzii (36%) and C. coccoides (22%) groups representing the predominant bacteria. No effect of the Lactobacillus strain supplementation was observed in the composition of the cecal microbiota assessed by fluorescent in situ hybridization with the 7 probes. Nevertheless, profiling of the cecal microbiota using temporal temperature gradient gel electrophoresis in combination with principal component analysis demonstrated an impact of the probiotic treatment on the overall bacterial community as well as on the Lactobacillus population.     

In Situ Hybridization for the Detection of Infectious Laryngotracheitis Virus in Sections of Trachea from Experimentally Infected Chickens

An in situ hybridization procedure for the detection of infectious laryngotracheitis virus (ILTV) in experimentally infected chickens is described. Formalin-fixed, paraffin-embedded sections of trachea, taken from chickens on days 3–10 post-inoculation (p.i.) with ILTV were hybridized with a mixture of 2 biotinylated, polymerase chain reaction-generated DNA fragments. The fragments correspond to sequences of the ILTV glycoprotein C and thymidine kinase genes. In situ hybridization was seen in 7 out of 7 chickens examined on day 3 p.i., 2 out of 2 examined on day 4 p.i. and 3 out of 3 examined on day 5 p.i. No hybridization was observed in 3 out of 3 chickens examined on day 10 p.i. ILTV nucleic acid was detected in nuclei of degenerated tracheal epithelial cells and in intranuclear inclusion bodies of syncytia.     

DNA fingerprints of farm animals generated by microsatellite and minisatellite DNA probes

A multi-locus DNA probe, R18.1, derived from a bovine genomic library, detected DNA fingerprints of highly polymorphic loci in hybridization to genomic DNA from poultry and sheep, and of moderate polymorphic loci in cattle and human DNA. The average numbers of detected bands in chickens and sheep were 27.8 and 21.4, and the average band sharing levels were 0.25 and 0.33, respectively. In hybridization to cattle and human DNA, the results were less polymorphic; nevertheless, individual identification is feasible using probe R18.1. The results obtained by R18.1 were compared to results obtained by Jeffreys minisatellite probe 33.6 and two microsatellite oligonucleotides, (GT)12 and (GTG)5. The total number of detected loci using probes R18.1 and 33.6 were estimated in chickens through family analysis of broilers and the maximal number of detectable loci was calculated.     

Acquisition of viral DNA sequences in target organs of chickens infected with avian myeloblastosis virus.

The distribution of oncornavirus DNA sequences in various tissues of normal chickens and of chickens with leukemia or kidney tumors induced by avian myeloblastosis virus (AMV) was analyzed by DNA-RNA hybridization using 35S AMV RNA as a probe. All the tissues from normal chickens which were tested contained the same average cellular concentration of endogenous oncornavirus DNA. In contrast, different tissues from lekemic chickens and from chickens bearing kidney tumors contained different concentrations of AMV homologous DNA: in some tissues there was no increase whereas other tissues acquired additional AMV-specific DNA sequences. The increase was the greatest in tissues which can become neoplastic after infection, such as myeloblasts, erythrocytes, and kidney cells. It was directly demonstrated that DNA from AMV-induced kidney tumor contains AMV sequences which are absent in DNA from normal cells. A similar finding had been previously obtained with leukemic cells (15). 3H-labeled 35S RNA from purified AMV was exhaustively hybridized with an excess of normal chicken DNA to remove all the viral RNA sequences which are complementary to DNA from uninfected cells. The 3H-labeled RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded RNA. The residual RNA hybridized to chicken kidney tumor DNA but did not rehybridize with normal chicken DNA.     

Sex chromosome linkage of chicken and duck type I interferon genes: further evidence of evolutionary conservation of the Z chromosome in birds

Type I interferons (IFNs) are a family of proteins that are predominantly expressed in response to viral infection. Two serologically distinct forms of type I IFN, designated ChIFN1 and ChIFN2, have recently been recognized in the chicken. ChIFN1 is encoded by a cluster of ten or more intronless genes, whereas ChIFN2, whose primary sequence is 57% identical, is encoded by a single intronless gene. By fluorescence in situ hybridization we now demonstrate that the genes for ChIFN1 and ChIFN2 are all located on the short arm of the chicken Z chromosome. This assignment was confirmed by results that showed that DNA from male (ZZ) chickens yielded approximately twofold stronger Southern blot signals with ChIFN1 and ChIFN2 hybridization probes than DNA from females (ZW). Attempts to determine differences in IFN production between male and female chickens failed owing to a high degree of variation in virus-induced IFN expression between individuals of both sexes. Sex linkage of IFN genes was also observed in domestic ducks: fluorescence in situ hybridization of duck metaphase chromosomes with a duck type I IFN probe was confined to the terminal region of the long arm of the Z chromosome. Thus, in contrast to mammals, which have their IFN genes on autosomes, birds have the type I IFN genes on the sex chromosome. Received: 9 January 1998     

Broad dissemination of Histomonas meleagridis determined by the detection of nucleic acid in different organs after experimental infection of turkeys and specified pathogen-free chickens using a mono-eukaryotic culture of the parasite

Histomonas meleagridis, a flagellated protozoan parasite, is the causative agent of histomonosis (syn. histomoniasis, blackhead) in turkeys and chickens. The organs primarily affected by the parasite are the caeca and the liver. Until now, only few reports exist in which the parasite has been diagnosed in tissues other than those mentioned above. Hence, the aim of this study was to perform a systematic investigation of various organs of turkeys and specified pathogen-free chickens following an experimental infection with a mono-eukaryotic culture of Histomonas meleagridis in order to determine the dissemination of the flagellate in infected birds. Molecular methods like PCR and in situ hybridization were used for this purpose. For the first time, the DNA of the parasite could be detected in 13 different organs of infected turkeys by PCR including the proventriculus, duodenum, jejunum, caeca, pancreas, bursa of Fabricius, liver, kidney, spleen, heart, lung, thymus and the brain. Most of these findings were further confirmed by in situ hybridization. In contrast to the turkeys that all died shortly after the infection, all of the chickens survived without displaying any clinical symptoms. Even at necropsy, only mild pathological changes were observed in the caeca. Nevertheless, the parasite could also be detected in various organs of these birds, namely the caeca, bursa of Fabricius, kidney, heart and the brain.     

Enterocyte expression of calbindin,calbindin mRNA and calcium transport increases in jejunal tissue during onset of egg production in the fowl (Gallus domesticus)

• 1.1. Quantitative measurements of calbindin mRNA, calbindin protein and calcium uptake have been made in sectioned intestinal villi to determine the location and cellular characteristics of their expression in immature, point of lay and laying chickens.
• 2.2. Trace amounts of calbindin mRNA were detected by in situ hybridization in enterocytes located around the crypt-villus junction in jejunal tissue taken from immature and point of lay chickens. Large amounts of calbindin mRNA were detected in upper crypt and all villus enterocytes in tissue taken from laying chickens. Maximal levels of calbindin mRNA occurred in the basal third of the villus in laying chickens.
• 3.3. No calbindin was detected immunocytochemically in tissue taken from immature and point of lay chickens. Large amounts of calbindin were expressed in tissue taken from laying chickens. Maximal expression of calbindin in this case occurred in villus tip enterocytes.
• 4.4. Rapid uptake of calcium by tissue taken from laying chickens was twice that found in immature and point of lay birds. Calcium uptake in tissue taken from laying hens was also shown by quantitative autoradiography to take place maximally in villus tip enterocytes.
• 5.5. Regulation ofcalbindin gene expression and the cellular characteristics of calcium transport in laying chickens are discussed in terms of an adaptive response taking place in birds undergoing a daily loss of egg shell calcium.

     

Excessive Cytokine Response to Rapid Proliferation of Highly Pathogenic Avian Influenza Viruses Leads to Fatal Systemic Capillary Leakage in Chickens

Highly pathogenic avian influenza viruses (HPAIVs) cause lethal infection in chickens. Severe cases of HPAIV infections have been also reported in mammals, including humans. In both mammals and birds, the relationship between host cytokine response to the infection with HPAIVs and lethal outcome has not been well understood. In the present study, the highly pathogenic avian influenza viruses A/turkey/Italy/4580/1999 (H7N1) (Ty/Italy) and A/chicken/Netherlands/2586/2003 (H7N7) (Ck/NL) and the low pathogenic avian influenza virus (LPAIV) A/chicken/Ibaraki/1/2005 (H5N2) (Ck/Ibaraki) were intranasally inoculated into chickens. Ty/Italy replicated more extensively than Ck/NL in systemic tissues of the chickens, especially in the brain, and induced excessive mRNA expression of inflammatory and antiviral cytokines (IFN-γ, IL-1β, IL-6, and IFN-α) in proportion to its proliferation. Using in situ hybridization, IL-6 mRNA was detected mainly in microglial nodules in the brain of the chickens infected with Ty/Italy. Capillary leakage assessed by Evans blue staining was observed in multiple organs, especially in the brains of the chickens infected with Ty/Italy, and was not observed in those infected with Ck/NL. In contrast, LPAIV caused only local infection in the chickens, with neither apparent cytokine expression nor capillary leakage in any tissue of the chickens. The present results indicate that an excessive cytokine response is induced by rapid and extensive proliferation of HPAIV and causes fatal multiple organ failure in chickens.     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.     

Distribution of Deoxyribonucleic Acid Complementary to the Ribonucleic Acid of Avian Myeloblastosis Virus in Tissues of Normal and Tumor-Bearing Chickens

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

运用套式PCR检测传染性喉气管炎病毒核酸

选择鸡传染性喉气管炎病毒保守TK基因的蛋白编码区域,设计并合成了一对外引物和一对内引物,建立并优化了检测鸡传染性喉气管炎病毒DNA的套式PCR法。通过检测ILTV感染的鸡胚绒毛尿囊膜,实验室病料和临床病料,结果表明,套式PCR法能检测出ILTV感染后的非免疫鸡胚和SPF鸡绒毛膜研磨液中的被稀释了10^5倍的病毒（约1fg的ILTV DNA）,攻毒后第10天还能从非免疫鸡和SPF鸡气管拭子中检出ILTV,第10天非免疫鸡气管拭子中ILTV的最大检出率为7／10,第10天SPF鸡气管拭子中ILTV的最大检出率为8／10。对非免疫鸡和SPF鸡的气管拭中ILTV最佳检出时间均在攻毒后第5天。对临床样品中的ILTV的最大检出率为7／7。经过核酸杂交验证,套式PCR法具有很高的特异性和敏感性,为从分子水平探讨ILTV的发病机理、临床早期快速诊断提供了新的研究手段。     

Assessment of Inbreeding by DNA Fingerprinting: Development of a Calibration Curve Using Defined Strains of Chickens

By analyzing DNA fingerprints of chickens from seven well-defined genetic groups, a calibration curve was established relating the degree of inbreeding with the average band frequency, allelic frequency and band sharing. The probe used was bacteriophage M13 DNA and digestion of the genomic DNA was carried out with the MspI restriction enzyme. The analysis also provided an estimate of the average allelic frequency at a hypervariable locus and the average mutation frequency per locus and generation. The values of 0.24 and 1.7 X 10(-3), respectively, are similar to the estimates for humans using other probes and hybridization protocols. It is suggested that the calibration curve established can be used for determining inbreeding not only in chickens, but also in other species.     

Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus)

In our previous studies, we demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, to investigate the differentiation pattern of female PGCs in male gonads in chickens, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W-bearing spermatogonia, spermatocytes, and round spermatids accounted for 30.8%, 32.7%, and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second meiotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis.     

Probable congenital transmission of reticuloendotheliosis virus caused by vaccination with contaminated vaccines

Contaminated vaccine is one unexpected and potential origin of virus infection. In order to investigate the most likely cause of disease in a broiler breeder company of Shandong Province, all 17 batches of live-virus vaccines used in the affected flocks and 478 tissue samples were tested by dot-blot hybridization, nested PCR, and IFA. The results suggested the outbreak of disease was most probably due to the vaccination of REV-contaminated MD-CVI988/Rispens vaccines and ND-LaSota+IB-H120 vaccines. Furthermore, the REV was probably transmitted to the commercial chickens through congenital transmission.     

Incidence of the aerobactin iron uptake system among Escherichia coli isolates from infections of farm animals

To assess the importance of aerobactin-mediated iron uptake as a bacterial virulence determinant in animal infections, a total of 576 strains of Escherichia coli isolated from cattle, chickens, sheep and pigs were screened by colony hybridization to determine the presence of the aerobactin genetic determinants, and by a bioassay to detect aerobactin secretion in iron-limited conditions. Results obtained by the two complementary methods correlated well. The incidence of the aerobactin system was very high among septicaemia isolates, particularly those from cattle and chickens, an observation that strongly suggests an important role for this mechanism of iron assimilation in pathogenesis. On the other hand, the incidence of the aerobactin system among mastitis strains was not significantly higher than among faecal isolates from healthy animals. No classical enterotoxigenic E. coli strains tested carried the aerobactin genetic determinants. Although most strains that produced aerobactin were also able to make colicin V, the fact that the two characteristics existed separately in a significant minority of isolates suggested that colicin testing alone could not be reliably used to determine the presence of the aerobactin system.     

Testing homology of loci for two plumage colors, "lavender" and "recessive white," with chicken and Japanese quail hybrids

Homology for two plumage color loci was studied by hybridization between chickens and Japanese quail. First, chicken-quail hybrids were produced from homozygous "lavender" chicken cocks and "bleu" Japanese quail, and all 30 hybrids had the same parental slate blue plumage color. On the other hand, no hybrids with this plumage were obtained out of 18 progeny from the same cocks and wild-type quail. These results show that the slate blue plumage color is determined by homologous loci in Japanese quail and chickens. Second, all (n = 25) chicken-quail hybrids hatched from homozygous "recessive white" cocks and "recessive white" (n = 8) or "wild-type" (n = 17) quail had the same pattern of plumage color, with white feathers on the ventral face and colored feathers elsewhere. These results indicate that the recessive white mutations are not homologous in Japanese quail and chickens.     

B cell maturation in the chicken Harderian gland

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.