Resistance to malignant catarrhal fever in American bison (Bison bison) is associated with MHC class IIa polymorphisms.

The Rhadinovirus ovine herpesvirus-2 (OvHV-2) is the most common causative agent of malignant catarrhal fever (MCF) in clinically susceptible ruminants including cattle and bison. American bison (Bison bison) are highly susceptible to clinical MCF. Nevertheless, approximately 20% of bison on ranches or in feedlots become infected with the virus without developing clinical disease. Defining the genetic basis for differences in susceptibility between bison could facilitate development of improved control strategies for MCF. One genetic region that influences susceptibility to infectious diseases is the major histocompatibility complex (MHC). In this study, a Bison bison (Bibi) DRB3 oligonucleotide microarray was used to type 189 bison from 10 herds where MCF outbreaks had occurred. Binary logistic regression was used to classify DRB3 alleles as resistant (R), susceptible (S) or neutral (N). Animals were reclassified using six DRB3 genotype categories: N/N, N/R, N/S, R/S, R/R and S/S. Analysis of homogeneity across herds showed that there was a herd effect. Consequently, a penalized logistic regression model was run with herd and genotype categories as the explanatory variables. The R/R genotype was associated with resistance to MCF (P = 0.0327), while the S/S genotype was associated with clinical MCF (P = 0.0069). This is the first evidence that MHC class IIa polymorphism is associated with resistance or susceptibility to OvHV-2-induced MCF.

Why is the fetal allograft not rejected?

In viviparous species, the conceptus must be protected from a potentially hostile maternal immune system. The major histocompatibility complex (MHC) is a genetic region that encodes MHC class I and class II proteins, which present peptide antigens to T lymphocytes and induce graft rejection. The MHC, class II proteins are only expressed on professional, antigen-presenting cells. However, classical, MHC class I proteins are expressed on all nucleated somatic cells. Protection of the conceptus from immune-mediated rejection involves downregulation of classical MHC class I antigen expression on trophoblast cells, which form the external epithelial layer of the placenta, and maintenance of an immunologically favorable immunosuppressive environment in the uterus. Normally, bovine trophoblast cells do not express MHC class I antigens before d 120 of pregnancy. However, during the last third of gestation, trophoblast cells in the inter-placentomal and arcade regions of the placenta express classical, MHC class I proteins, which could potentially induce fetal rejection, as well as nonclassical, MHC class I proteins. A human, nonclassical, MHC class I antigen, human leukocyte antigen G, is an important immunoregulatory factor required for the maintenance of pregnancy. In cattle, MHC class I expression during the last third of pregnancy has no adverse effects and probably contributes to placental separation at parturition. However, somatic cell nuclear transfer (SCNT) conceptuses, the majority of which are aborted between d 30 and 90 of pregnancy, had trophoblast cell expression of MHC class I antigens before d 34 of pregnancy. In conjunction with increased trophoblast MHC class I expression, SCNT pregnancies exhibited a marked increase in the number of stromal lymphocytes in the uteri of surrogate dams. A retrospective study found that SCNT pregnancies established using MHC class I-homozygous cell lines, in which the immunological barrier is greatly reduced, had significantly improved fetal survival from d 28 to term (51% survival for MHC-homozygous and 5% for MHC-heterozygous SCNT fetuses). Consequently, it appears that the high rate of fetal mortality in SCNT pregnancies is due, at least in part, to inappropriate expression of trophoblast, MHC class I antigens resulting in immune-mediated placental rejection. This suggests that appropriate regulation of MHC class I genes is critical for immunological acceptance of an allogeneic conceptus.

Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows.

In ruminants, pregnancy results in up-regulation of a large number of IFN-stimulated genes (ISG) in the uterus. Recently, one of these genes was also shown to increase in peripheral blood leukocytes (PBL) during early pregnancy in sheep. Our working hypothesis is that conceptus signaling activates maternal gene expression in PBL in dairy cattle. The objectives of this study were to characterize ISG expression in PBL from pregnant (n = 20) and bred, nonpregnant (n = 30) dairy cows. Steady-state levels of mRNA for Mx1, Mx2, beta2-microglobulin, ISG-15, IFN regulatory factor-1, and IFN regulatory factor-2 were quantified. Holstein cows were synchronized to estrus and artificially inseminated (d 0). Blood samples were collected (coccygeal venipuncture) on d 0 and 16, 18, and 20 d after insemination for progesterone analysis and PBL isolation. Pregnancy was confirmed by transrectal ultrasonography at approximately 40 d after breeding. A status x day interaction was detected for Mx1, Mx2, and ISG-15 gene expression. When analyzed within day, levels of mRNA for ISG-15 and Mx1 were greater in pregnant compared with bred, nonpregnant cows on d 18 and 20, respectively. Expression of the Mx2 gene increased in the pregnant group compared with bred, nonpregnant cows on d 16, 18, and 20 after insemination. beta2-Microglobulin, IFN regulatory factor-1, and IFN regulatory factor-2 were not different between groups. The results clearly indicated that components of the innate immune response are activated in PBL during the period of pregnancy recognition and early embryo signaling. The physiological implications of these changes on maternal immune function are as yet unknown; however, they do provide a unique opportunity to identify bred, nonpregnant, cows 18 d after insemination in dairy cattle.

Major histocompatibility antigen expression on the bovine placenta: its relationship to abnormal pregnancies and retained placenta.

In viviparous animals, regulation of expression of major histocompatibility complex (MHC) class I antigens by the trophoblast cells, which constitute the outermost layer of the placenta, seems to be critical for maternal immunological acceptance of an allogeneic fetus. Cattle are unusual in this regard, since the bovine trophoblast cells, in specific regions of the uterine/placental interface, normally express MHC class I antigens during the third trimester of gestation. This expression appears to be biologically relevant as MHC class I compatibility between a cow and her fetus has been associated with an increased incidence of placental retention. We have found significant differences in lymphocyte populations, cytokine production, and trophoblast cell apoptosis in the placentomes of MHC-compatible and -incompatible pregnancies at parturition. This suggests that maternal immunological recognition of fetal MHC class I proteins triggers an immune/inflammatory response that contributes to placental separation at parturition in cattle. Early in pregnancy, a complete shutdown of MHC class I expression by trophoblast cells appears to be critical for normal placental development and fetal survival. In bovine somatic cell nuclear transfer (SCNT) pregnancies, there is an extremely high rate of fetal loss between days 30 and 90 of pregnancy. We have shown that in bovine SCNT pregnancies, between days 34 and 63 of gestation, there is both abnormal expression of MHC class I antigens by trophoblast cells and an abnormal accumulation of lymphocytes within the uterine stroma. Consequently, it is likely that activation of the maternal mucosal immune system, within the uterus at the same time when placentomes are being established, interferes with the process of placentome development and leads to immune-mediated abortion. Our data suggest that bovine MHC-compatible pregnancies provide a unique model for studying regulation of the uterine immune system, as well as immune-mediated placental rejection.

Long line positioning in neonates: does computed radiography improve visibility?

OBJECTIVES: To assess the use of soft copy reporting of computed radiography (CR) images in determining intravenous long line tip position in neonates and compare visibility rates with hard copy printed images. METHOD: A retrospective study of all long lines inserted on the neonatal unit over a period of one year was performed. Forty five lines were inserted in 30 neonates over this time. Assessment of the CR images was made by three independent observers by reviewing the films on the viewing console and as hard copy printed films. RESULTS: Accurate identification of the line tip could be made in 66.7% of cases (kappa = 0.9) using hard copy images and 95.6% cases (kappa = 1.0) using soft copy reporting (significant difference: p = 0.002). The difference in percentage visibility using the two techniques was 28.9% (95% confidence interval 10.2% to 36.7%). CONCLUSION: The use of soft copy review of CR image improves the visibility of the line tip position compared with hard copy films and reduces the need for repeat radiographs with/without intravenous contrast.

Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer.

In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

Splenogonadal fusion: report of a rare variety.

We report an unusual case of splenogonal fusion in a 10-year-old boy with an undescended left testis. He suffered from congenital limb defects, a known association with splenogonadal fusion, and had originally been admitted for orchidopexy.

The bovine placenta before and after birth: placental development and function in health and disease.

This paper reviews bovine placental development, anatomy (microscopic and gross), nomenclature and classification. The paper focuses on the biology of those specialized cells that arise from the outermost layer of very early embryos, the trophoblast cells, and on placental macrophages, cells that play a key role in fetal/placental defense. Data is presented from an immunohistochemical quantitative study that characterizes the ontogeny of placental macrophages using placental tissues from 21 cows (sampled from 4 months of pregnancy through the post partum period). Understanding of bovine placental development is essential for veterinarians, pathologists, diagnosticians and researchers. Lesions of diagnostic significance can be recognized for many economically important infectious abortifacient diseases, and there is growing evidence that pregnancy failure of cloned calves is due in part to unexplained placental failure. Placentology and placental pathology are becoming of increasing importance.

Temporal and regional regulation of major histocompatibility complex class I expression at the bovine uterine/placental interface.

In most mammals trophoblast cells do not express major histocompatibility complex (MHC) antigens. This probably protects the placenta from immune attack. We have used immunohistochemistry to study the ontogeny of MHC class I expression by bovine trophoblast and endometrial epithelial cells. The interplacentomal, placentomal arcade and placentomal villous/crypt regions were studied. In the interplacentomal region a substantial proportion of trophoblast cells were class I positive from the sixth month on and about half of the endometrial epithelium was class I positive throughout pregnancy. In the arcade region trophoblast class I expression was first observed in the sixth month, increased slowly and peaked at term. Here there was no endometrial epithelial class I expression until term and then only a small percentage of cells were positive. In contrast, in the placentomal villous/crypt region both trophoblast and endometrial epithelium were class I negative throughout gestation. This study shows that cattle have extensive trophoblast class I expression. Moreover class I expression on placentomal, cryptal endometrial epithelium is shut down. Because binucleate trophoblast cells migrate and fuse with endometrial epithelial cells, total shut down of class I expression in areas of intimate interdigitation may be critical for avoidance of immunological rejection.

A Hough transform for detecting the location and orientation of three-dimensional surfaces via color encoded spots.

Video-rate three-dimensional (3-D) acquisition is desirable, in particular for capturing the mouth's shape when modeling the vocal tract. In a new structured light technique, scenes are illuminated by an array of circular spots which are color encoded to resolve spatial ambiguity. The position and shape of the imaged spots depend on the location and orientation of the illuminated 3-D surface. We present a novel 3-D Hough transform (HT) to detect 3-D surface location and orientation via the imaged spots, with voting constraints applied to maximize potential accuracy. This new technique is demonstrated to successfully extract the 3-D data for a moving face from images acquired at video-rate.

PRIMO: A primer design program that applies base quality statistics for automated large-scale DNA sequencing.

PRIMO is a computer program that designs walking primers for large-scale DNA sequencing projects. Oligonucleotide primers are predicted automatically, using quality information associated with each base call, eliminating the need for manually viewing the sequence traces or inspecting contig assemblies to determine appropriate locations for primer design. This allows PRIMO to run in batch mode on an arbitrarily large number of templates. For shotgun sequencing, PRIMO reads assembled sequence contigs with corresponding base quality statistics and automatically designs walking primers as needed to extend and join contigs, or improve their overall quality. In the opposite extreme of single-pass or completely directed sequencing, PRIMO reads the unassembled sequence for each template and designs walking primers for extending each read. If the base-calling software does not provide base quality statistics, PRIMO assigns its own measure of base quality determined by the shapes of individual peaks in the trace data for each template. In this way, PRIMO can be used in the finishing stages of a shotgun sequencing project, in sequencing by directed primer walking, or in some intermediate strategy. The code is written in ANSI C and maintained in two versions: one for the Macintosh and the other for UNIX.

Insertion site specificity of the transposon Tn3.

The Tn3-deletion method [Davies and Hutchison, Nucleic Acids Res. 19, 5731-5738, (1991)] was used to sequence a 9.4 kb DNA fragment. Transpositional 'warm' spots were not a limiting factor but a 935 bp 'cold' spot was completed using a synthetic oligonucleotide primer. Two hundred and twenty three miniTn3 insertion sites from three sequencing projects were aligned and a 19 bp asymmetric consensus site was identified. There is no absolute sequence requirement at any position in this consensus, so insertion occurs promiscuously (approximately 37% of sites are potential targets). In our sequencing projects, multiply targeted sites always closely matched the consensus, although not all close matches were targeted frequently. The 935 bp cold spot showed no unusual features when analysed with the consensus sequence. The consensus can be used to accurately predict likely insertion sites in a new sequence. Synthetic oligonucleotides based on the consensus and a known hot spot for Tn3 were mutagenised. These sequences were not hot spots in our vectors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot. We conclude that some other factor, such as DNA secondary structure, also plays an important role in target site selection for the transposon Tn3.

Polymorphism of bovine MHC class I genes. Joint report of the Fifth International Bovine Lymphocyte Antigen (BoLA) Workshop, Interlaken, Switzerland, 1 August 1992.

The objectives of the Fifth International BoLA Workshop were to: standardize nomenclature, compare typing methods, and characterize BoLA haplotypes. The workshop was based on the distribution of blood samples (cells) from 60 selected cattle to 14 laboratories. Results for the class I (BoLA-A) region are presented in this paper while results for the class II regions are presented in a separate report. Thirty-six of the 50 previously established serological class I specificities were represented in the cell panel. However, only 30 specificities could be confirmed. Two specificities, A16 and A32, were upgraded from provisional, workshop (w) specificities to BoLA-A locus specificities and three new specificities, w51(w28), w52 and w53(w28), were defined. The 39 specificities distinguished 30 class I haplotypes in the 60 animals. Class I isoelectric focusing proved to be a useful adjunct to the serology. Isoelectric focusing confirmed several serologically defined splits and detected splits of A15(A8), A18(A6) and A22(w49) that had not been detected by serology. Subsequently, serological support for splits of A15(A8) and A22(w49) was found.

Polymorphism of bovine MHC class II genes. Joint report of the Fifth International Bovine Lymphocyte Antigen (BoLA) Workshop, Interlaken, Switzerland, 1 August 1992.

Polymorphism of the bovine DRB, DQA, DQB, DYA, DOB and DIB genes was investigated using restriction fragment length polymorphism (RFLP) analysis, isoelectric focusing (IEF), class II serology and polymerase chain reaction (PCR) based typing techniques. The simultaneous application of multiple typing techniques and the characterization of multiple genes resulted in a greatly enhanced picture of the bovine class II regions. Thirty-eight class IIa (DR-DQ) and 5 class IIb (DYA-DOB-DIB) haplotypes were defined. It was found that IEF types were associated with DRB3 polymorphism defined by DRB3 PCR-RFLP and DRB3 microsatellite PCR. Serologically defined polymorphism was associated with distinct molecular/IEF motifs and, therefore, DR and DQ specificities could be tentatively distinguished. Although the DR and DQ genes are tightly linked, neither DR nor DQ typing defined all of the class IIa region polymorphism. Furthermore, even the most powerful DRB3 typing technique, DRB3 PCR-RFLP, failed to detect all expressed DRB3 polymorphism. All detected DRB3 polymorphism could, however, be distinguished with a combination of two molecular techniques: DRB3 PCR-RFLP and DRB3 microsatellite PCR. RFLP typing with transmembrane probes detected significantly less polymorphism than typing with cDNA or exon probes. However, the transmembrane probes were useful because they were locus specific. The presence of only 5 of 12 possible class IIb haplotypes was unexpected and indicates that the DYA, DOB and DIB genes are tightly linked.

Serological definition of bovine MHC class II polymorphism in Holstein-Friesians.

Analysis of the reaction patterns of 57 putative class II MHC alloantisera revealed 18 B-cell serum clusters, named Ds01-Ds18. These clusters were grouped together in such a manner that 17 provisional serological class II haplotypes were defined. Segregation in half-sib families of these class II haplotypes with serologically defined class I and 1D-IEF defined DRB3 types indicated that the 18 clusters identify class II polymorphisms in cattle and can be considered as local specificities.

Strong association between polymorphisms in an intronic microsatellite and in the coding sequence of the BoLA-DRB3 gene: implications for microsatellite stability and PCR-based DRB3 typing.

A highly polymorphic microsatellite in the bovine DRB3 gene was characterized by polymerase chain reaction (PCR) analysis and DNA sequencing. A very strong association between expressed DRB3 polymorphism and microsatellite alleles was revealed by PCR analysis of genomic DNA from 116 animals representing three breeds of cattle. The results indicated a low frequency of microsatellite length mutations as the association was consistent over breeds. The DRB3 microsatellite may be utilized in a PCR-based typing method of bovine class II alleles. The microsatellite polymorphism did not distinguish all known DRB3 alleles, but it was shown that this method may be complemented by the use of allele-specific PCR based on the extensive polymorphism in the DRB3 exon 2. The DNA sequences of seven microsatellite alleles, associated with different class II haplotypes, were determined. The DRB3 microsatellite is composed of three repeat motifs, a stretch of at least 10 uninterrupted (TG)n dinucleotides, a long but interrupted stretch of (GA)n dinucleotides, and a few (CAGA)n tetranucleotides. There were pronounced sequence differences between alleles and the results indicated that the evolution of this microsatellite has involved length mutations of the dinucleotide repeats as well as point mutations causing interruptions in the dinucleotide repeats.

The microanatomy of the alveolar duct of the human lung imaged by confocal microscopy and visualised with computer-based 3D reconstruction.

The most widely accepted model of human lung alveolar duct systems is that they are constructed from central helical fibres between the turns of which lie alveolar opening. Experimental difficulties of handling and sectioning lung tissue have made it difficult to confirm this. Confocal laser scanning microscopy (CLSM) was therefore used to generate optical serial sections of the lung that were reconstructed in three dimensions and displayed using volume rendering techniques. From images of the reconstructions, a new structure is proposed in which alveolar ducts consist of collections of connected oval, twisted loop structures with eccentric openings.

Influence of host genetics upon antibody responses against gastrointestinal nematode infections in cattle.

Previous studies have shown that the number of gastrointestinal nematode eggs released per gram of feces (EPG) of calves is strongly influenced by host genetics. The purpose of this study was to determine if host genetics also influenced immune recognition of parasite antigens in these same calves. Serum samples were taken at monthly intervals from calves during their first grazing season, from approximately 4 months after the onset of calving and were continued until weaning. Serum samples were analyzed for antibodies against Ostertagia ostertagi, Haemonchus placei, Cooperia oncophora, and Oesophagostomum radiatum. Significant rises in antibodies of the IgG1 class were seen against Ostertagia ostertagi, H. placei, and C. oncophora. In addition, rises in anti-Ostertagia antibodies of the IgG2 and IgM isotypes were also noted. During periods of elevated antibody responses, the sire of the individual calves was found to influence significantly the level of circulating antibody. The heritability of serum anti-parasite antibody levels was demonstrated to be between 70 and 80%, depending upon the time and antibody isotype. The antibody levels did not appear to be correlated with parasite EPG values. These results indicate that the ability of calves to recognize parasite antigens is strongly influenced by genetic factors, and that the genetic factors which control antibody responses may differ from those controlling EPG values.

Characterization of bovine MHC class II polymorphism using three typing methods: serology, RFLP and IEF.

Various methods, with different strengths and weaknesses, are currently used to define polymorphism of the bovine major histocompatibility complex (MHC) class II genes. A more complete characterization of bovine lymphocyte antigen (BoLA) haplotypes can be achieved by combining several of these methods. In this study BoLA class II polymorphism was characterized using three typing methods: serology, restriction fragment length polymorphism (RFLP), and isoelectric focusing (IEF). Twenty six Holstein-Friesian and 15 Angus cattle that carried an array of serologically defined BoLA haplotypes were selected for the study. The panel included 12 BoLA complex homozygotes. The three class II typing methods recognized polymorphism associated with the same or very tightly linked genes in the DQ-DR class II subregion. In total 25 BoLA-A locus (class I)--DQ-DR subregion (class II) haplotypes were defined. Three of the serological class II specificities, Dx1, Dx3, and Dx4, were associated with more than one RFLP defined DQ-DR haplotype. The other 4 class II specificities behaved as private specificities. One BoLA haplotype was found in both Holstein and Angus cattle. Two other BoLA haplotypes defined here have previously been described in other breeds. This suggests that these haplotypes exist in strong linkage disequilibrium.