Establishment and initial characterization of continuous in vitro cultures of bovine T lymphocytes.

Utilizing human recombinant interleukin 2 (HrIL-2) in combination with several mitogens, continuously growing cultures of bovine lymphocytes have been established. Continuous presence of HrIL-2 is required to maintain the growth of these cultures but a requirement for continued presence of mitogen seems variable. Cloned lines have been derived from these cultures by limiting dilution and outgrowth in the presence of mitomycin C treated bulk cells. Cell surface phenotype analysis indicates that all of these cultures and cloned lines are of the T cell lineage. Functional analysis indicated that some of these cultured cells are cytolytically active in lectin mediated assays whereas others are not. Utilization of this technology for the growth and characterization of bovine T cells will undoubtedly contribute to an enhanced understanding of the bovine immune response mechanisms.

Establishment of equine T-lymphocyte cultures dependent on recombinant human interleukin-2.

Long-term equine lymphocyte cultures were initiated and maintained in continuous culture with medium containing recombinant human interleukin-2. Cultures were successfully maintained with lectin activation signals and recombinant human interleukin-2 or with recombinant human interleukin-2 alone. All cell cultures that were characterized had a T-lymphocyte phenotype and had lectin-dependent or -independent cytotoxicity directed to various cell types. These findings demonstrate that long-term equine T-lymphocytes cultures can be initiated and maintained easily.

Silent blood chimaerism in a mare confirmed by DNA marker analysis of hair bulbs.

Microsatellite DNA markers in a mare's hair bulbs not concordant with markers in her blood confirmed the hypothesis of chimaerism which had been proposed to explain the apparent parentage exclusion of the mare from her suckling foal. Parentage analysis for this foal based on genetic markers not originating from blood cells of its dam supported a parentage verification conclusion.

Interaction of bluetongue virus with bovine lymphocytes.

Freshly isolated, and established, cultures of bovine peripheral blood mononuclear leukocytes (PBMLs) were exposed to bluetongue virus (BTV) for the purpose of defining potential lymphotropism. PBML cultures were established in the presence of interleukin 2 (IL-2) and mitogen and maintained either as bulk culture or were cloned prior to infectivity studies. All cultures appeared to be of the T cell phenotype based on the following characteristics: binding of T lymphocyte-specific lectins (i.e. peanut agglutinin and Helix pomatia), rosetting of sheep erythrocytes, binding of a putative pan-T monoclonal antibody, and absence of surface immunoglobulin (Ig). T lymphocyte cultures were further characterized by their ability to elicit lectin-dependent cellular cytotoxicity (LDCC). Exposure of established lymphocyte cultures to BTV resulted in productive cytopathic and non-cytopathic infections. Non-cytopathic productive infections were observed in LDCC-negative cultures whereas cytopathic and non-cytopathic infections were observed in LDCC-positive cultures. Exposure of freshly isolated PBMLs to BTV resulted in minimal virus replication; addition of mitogen and IL-2 to such cultures did not augment virus replication. Addition of mitogen and IL-2 induced negligible blast transformation, whereas PBML viability was minimally affected. These studies establish a tropism of BTV for bovine T lymphocytes with virus replication being limited to those cells undergoing blastogenesis. Establishment of infected lymphocyte cultures, without loss of culture viability, suggest such an interaction may contribute to the long term viraemias associated with BTV infection of cattle.

Integrated bovine leukosis proviral DNA in T helper and T cytotoxic/suppressor lymphocytes.

Bovine leukosis virus (BLV) is associated with the disease complex enzootic bovine leukosis. The infection may remain clinically silent in the form of an aleukaemic state or emerge as a persistent lymphocytosis and more rarely as lymphosarcroma. BLV has been considered classically to be a B lymphotropic virus, based upon the absolute increase in B lymphocytes in persistent lymphocytosis, the B lymphocyte phenotype of a majority of the cells making up lymphosarcomas and the identification of viral antigen expressed in B lymphocytes following in vitro culture of peripheral blood mononuclear leukocytes. This association of BLV with B lymphocytes is well established but the mechanism(s) of disease expression is not defined. To examine further the cellular tropism(s) of BLV, T lymphocyte subpopulations from 10 lymphocytotic cattle were established in vitro. Lymphocyte cultures were characterized by their subpopulation phenotype and DNA was extracted for identification of integrated provirus by Southern blot hybridization. Provirus was identified in T lymphocyte cultures derived from seven of 10 lymphocytotic cattle, with both T helper and T cytotoxic/suppressor subpopulations affected.

A single base transversion in the flanking region of an equine microsatellite locus affects amplification of one allele.

The equine dinucleotide microsatellite HMS7 is part of a microsatellite panel utilized in a parentage verification programme at the Veterinary Genetics Laboratory (Davis, California, USA). Apparent non-Mendelian inheritance was noted when a Quarter Horse mare was excluded as the parent of two offspring based on analysis of the HMS7 locus. The mare's DNA type qualified her as a parent of the offspring at an additional 20 microsatellite loci. The three animals appeared homozygous for HMS7 with each possessing an allele different from that of the other two animals. Polymerase chain reaction primers designed to bind outside the published primer-binding sites amplified an additional shared allele in all three horses, which qualified the mare as the dam of the two offspring. Sequencing of this newly detected allele revealed a C to A transversion in one of the published primer-binding regions. Apparent non-Mendelian inheritance at the HMS7 locus has been encountered in an additional 26 Quarter Horse parentage cases. In all instances, the lack of amplification and resultant 'null' allele was shown to be caused by the same transversion.

Validation of microsatellite markers for routine horse parentage testing.

A parallel testing of 4803 routine Quarter Horse parentage cases, using 15 loci of blood group and protein polymorphisms (blood typing) and 11 loci of dinucleotide repeat microsatellites (DNA typing), validated DNA markers for horse pedigree verification. For the 26 loci, taken together, the theoretical effectiveness of detecting incorrect parentage was 99.999%, making it extremely unlikely that false parentage would fail to be recognized. The tests identified incorrect parentage assignment for 95 offspring (2% of cases). Despite fewer loci, DNA typing was as effective as blood typing and, in parentage exclusion cases, provided more systems to substantiate the genetic incompatibility. Five offspring presented potential genetic incompatibilities with their parents in only a single microsatellite system, but the parentage exclusions could not be confirmed with discordant results at additional loci. Two of these five incompatibilities could be explained as consequences of a null allele and three as fragment size increases or decreases (putative mutations). Provided that an exclusion assignment was based on at least two systems of genetic incompatibility, such rare genetic events did not lead to false exclusions. Notwithstanding the near 100% effectiveness estimations for either typing panel alone to identify incorrect parentage, this validation test showed an actual effectiveness of 97.3% for blood typing and 98.2% for DNA typing. The DNA-based test, however, may feasibly achieve higher efficacy than reported here by adding selected systems to the parentage test panel.