Christopher J. Davies

Abnormal Expression of Trophoblast Major Histocompatibility Complex Class I Antigens in Cloned Bovine Pregnancies Is Associated with a Pronounced Endometrial Lymphocytic Response

Abstract Early embryonic losses are much higher in nuclear transfer (cloned) pregnancies, and this is a major impediment to improving the efficiency of cloned animal production. In cattle, many of these losses occur around the time of placental attachment from the fourth week of gestation. We studied the potential for altered immunologic status of cloned pregnancies to be a contributing factor to these embryonic losses. Expression of major histocompatibility complex class I (MHC-I) by trophoblast cells and distribution of endometrial T-lymphocyte numbers were investigated. Six 5-wk-old cloned pregnancies were generated, and 2 others at 7 and 9 wk were also included, all derived from the same fetal cell line. All 8 cloned placentas displayed trophoblast MHC-I expression. None of the 8 controls (4–7 wk old) showed any MHC-I expression. The percentage of trophoblast cells expressing MHC-I varied in the clones from 17.9% to 56.5%. Numbers of T lymphocytes (CD3+ lymphocytes) were significantly higher in the endometrium of the majority of cloned pregnancies compared with controls. In the cloned pregnancies, large aggregates of T cells were frequently observed in the endometrium in addition to increased numbers of diffusely spread subepithelial lymphocytes. As trophoblast MHC-I expression is normally suppressed during early gestation, the observed MHC-I expression in the cloned pregnancies is likely to have induced a maternal lymphocytic response that would be detrimental to maintaining viability of the cloned pregnancy. These findings support a role for immunologic rejection in the syndrome of early embryonic loss in cloned bovine pregnancies.

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions.

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DOβ, DQα, DQβ, DRαand DRβgenes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZα, DYα, and DYβ, are reported. DZαwas assumed to correspond to the human DZαgene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQα, DQβ, DRαand DRβloci and the other one is composed of the DOβDYα, DYβ, and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational “hot spot” in the bovine class II region.

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.

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Göttingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.

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.

POLYMORPHISM OF BOVINE MHC CLASS II 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.

Analysis of the Immune Response to Mycobacterium avium subsp. paratuberculosis in Experimentally Infected Calves

ABSTRACT Johnes disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4+ T cells with a memory phenotype (CD45R0+) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8+ T cells proliferated in response to antigens until 18 months p.i. γδ T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1+ CD2− and a few WC1− CD2+ γδ T cells expressed CD25 at time zero. By 18 months, however, subsets of γδ T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3− non-T non-B null cells, CD2+ and CD2−, proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.

Neospora caninum-Infected Cattle Develop Parasite-Specific CD4+ Cytotoxic T Lymphocytes

ABSTRACT Cattle infected with Neospora caninum readily experience transplacental parasite transmission, presumably after maternal parasitemia, leading to abortion or birth of congenitally infected calves. Cytotoxic T lymphocytes (CTL) are important mediators of protective immunity against Toxoplasma gondii, an intracellular apicomplexan protozoan closely related to N. caninum. In this study, N. caninum-specific CTL expanded from peripheral blood mononuclear cells of two major histocompatibility complex-mismatched, experimentally infected cattle were identified by using a 51Cr release cytotoxicity assay. Enrichment and blocking of CD4+- and CD8+-T-lymphocyte effector subsets indicated that CD4+ CTL killed N. caninum-infected, autologous target cells and that killing was mediated through a perforin/granzyme pathway. Detection and characterization of CTL responses to N. caninum in the natural, outbred, bovine host will facilitate identification of immunogens and design of immunization strategies to induce parasite-specific CTL against transplacental N. caninum transmission in cattle.

Evidence for expression of both classical and non-classical major histocompatibility complex class I genes in bovine trophoblast cells

During the third trimester of pregnancy bovine trophoblast cells in the interplacentomal and arcade regions of the placenta express major histocompatibility complex class I (MHC‐I) antigens. At parturition immunological recognition of MHC‐I antigens appears to contribute to normal placental release. Therefore, we hypothesized that during late pregnancy bovine trophoblast cells express polymorphic, classical MHC‐I antigens.

Genetic control of immunity to gastrointestinal nematodes of cattle.

Previous studies have indicated that host genetics significantly affects the number of gastrointestinal nematode eggs per gram (epg) in the feces of calves during their first grazing season. An entire calf crop of approximately 190 animals was monitored monthly until weaning to verify these earlier results, and to begin to discern the basis for this phenomenon. A significant genetic effect on fecal epg values was not observed until calves had been on pasture for 2-3 months, and was demonstrable until late in the grazing season when the effect was lost. The loss of a genetic effect coincided with the appearance of significant numbers of the more highly fecund nematode species Haemonchus placei and Oesophagostomum radiatum, and with an apparent increase in Ostertagia ostertagi transmission, indicating that the observed genetic control of epg values may be species specific, dose dependent or both. Calves were selected from the population, and grouped according to their epg phenotype over the grazing season as either high or low epg calves. Postmortem examination of some of these calves indicated that worm burdens in the low epg calves were 60% of those of the high epg calves. Experimental challenge inoculation of the remaining calves indicated that: (1) challenge with Cooperia oncophora resulted in low epg calves harboring worm numbers that were 65% of those of high epg calves; (2) challenge with O. ostertagi resulted in similar numbers of worms in both groups, but the fecundity of worms in the low epg groups was significantly lower (P less than 0.05) than in the high epg group. Analysis of serum anti-Ostertagia antibody levels in the grazing calf population showed rises in serum IgG1, IgG2, IgM and IgA antibody levels during the grazing season. Peak serum IgG2 and IgG1 anti-Ostertagia antibody levels were found to be significantly affected by host genetic factors while IgA and IgM levels were not under such control.