Cytokine gene expression at the materno-foetal interface after experimental Neospora caninum infection of heifers at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle, but the reasons why only some animals abort remain unclear. The immunological control of the parasite in the placenta or by the foetus could be the key to determining the mechanism of abortion and/or transplacental transmission to the foetus. In this study, cytokine gene expression, analysed by real-time RT-PCR, at the maternal (caruncle) and foetal placenta (cotyledon) of heifers infected at 110 days of gestation by intravenous inoculation of N. caninum tachyzoites was compared with the responses in uninfected heifers. Animals were euthanized 3 weeks after infection. Upregulated Th1, Th2 and T-regulatory (Treg) cytokine gene expression was observed in both the maternal and the foetal placenta in the infected group. In the caruncle of infected animals, the main changes included upregulation of IFN-γ, IL-12p40, IL-6 and IL-10. In the cotyledon, the main changes included upregulation of IFN-γ and downregulation of TGF-ß, being the later the only cytokine downregulated in the infected group. The observed cytokine expression pattern was associated with alive but transplacentally infected foetuses, suggesting that such cytokine pattern is beneficial to foetal survival, but could have a role in the transplacental transmission of the parasite.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Detection of germline and somatic copy number variations in cattle.

As a complement to the Bovine HapMap Consortium project, we initiated a systematic study of the copy numbervariation (CNV) within the same cattle population using array comparative genomic hybridization (array CGH). Oligonucleotide CGH arrays were designed and fabricated to cover all chromosomes with an average interval of 6 kb using the latest bovine genome assembly. In the initial screening, three Holstein bulls were selected to represent major paternal lineages of the Holstein breed with some maternal linkages between these lines. Dual-label hybridizations were performed using either Hereford L1 Dominette 01449 or L1 Domino 99375 as reference. The CNVs were represented by gains and losses of normalized fluorescence intensities relative to the reference. The data presented here, for the first time, demonstrated that significant amounts of germline and fewer somatic CNVs exist in cattle, that many CNVs are common both across diverse cattle breeds and among individuals within a breed, and that array CGH is an effective tool to systematically detect bovine CNV. Selected CNVs have been confirmed by independent methods using real-time (RT) PCR. The strategy used in this study, based on genome higher-orderarchitecture variation, is a powerful approach to generating resources for the identification of novel genomic variation and candidate genes for economically important traits.

Enzymatic amplification and molecular cloning of cDNA encoding the small and large subunits of bovine interleukin 12.

cDNA generated from stimulated abomasal lymph node cells was used to amplify and clone the 35 kDa and 40 kDa subunits of bovine interleukin 12 (IL-12) using primers derived from semi-conserved regions between human and mouse IL-12 sequences. The deduced amino acid sequence of the 40 kDa subunit demonstrated 84.4% and 67.6% homology with human and mouse sequences, respectively. The deduced sequence of the 35 kDa subunit exhibited comparable similarities to the human 35 kDa subunit (82.2%) but differed significantly (58.6%) from mouse-derived sequences.

Molecular cloning of cDNA encoding porcine interleukin-15.

Interleukin-15 (IL-15) is a recently identified growth and differentiation factor with an important potential role in the initial immune responses to infection. To enable the study of the role of this cytokine in the protective immune-mechanisms generated against parasitic diseases of swine, cDNA was generated from a macrophage enriched adherent cell population from peripheral blood mononuclear cells (PBMC). This cDNA was used for the enzymatic amplification of the porcine IL-15 sequence using human IL-15-derived primers. The open-reading frame of the porcine IL-15 cDNA is 486 base pairs (bp) in length and encodes a 162-amino-acid (aa) protein. Comparisons of the predicted swine protein sequence with those predicted from human, bovine and mouse IL-15 sequences indicate similarities of 82.1, 84.6, and 71.6%, respectively.

Cloning and expression of bovine interleukin-15: analysis and modulation of transcription by exogenous stimulation.

The bovine interleukin-15 (IL-15) sequence was cloned from abomasal lymph node mRNA by enzymatic amplification of cDNA using human primers proximal to and including the translation start and stop sites. The open reading frame is 486 base pairs in length, and the proposed protein sequence shows 78.4% and 73.5% similarity with that predicted for the human and mouse sequences, respectively. Expressed and purified recombinant bovine IL-15 in the absence of the 48-amino acid leader sequence stimulated the proliferation of bovine lymphoblast cells at least 12-fold over background at maximum concentration levels. Competitive reverse transcriptase-polymerase chain reaction analysis showed constitutive levels of IL-15 mRNA within a broad range of tissues and cell types. Lipopolysaccharide addition to adherent lymph node populations caused moderate increases in IL-15 transcription, whereas the addition of phorbol 12-myristate 13-acetate and calcium ionophore failed to induce gene expression for this cytokine. Transcription of IL-15 was also downregulated in the presence of low concentrations of human recombinant interleukin-2.

Immunization with Babesia bigemina rhoptry-associated protein 1 induces a type 1 cytokine response.

Rhoptry-associated protein-1 (RAP-1) homologues of Babesia bigemina and Babesia bovis are promising candidates for inclusion in subunit vaccines against these hemoprotozoan parasites. Partial protection against challenge infection has been achieved with native forms of these antigens, but the mechanism of immunity has not been thoroughly defined. We previously demonstrated that a panel of antigen-specific T helper cell clones derived from B. bigemina RAP-1-immunized cattle expressed relatively high levels of interferon-gamma (IFN-gamma) protein and transcript and low levels of interleukin-4 (IL-4), indicative of a type 1 immune response. In the current study we present evidence that subcutaneous immunization with native B. bigemina RAP-1 protein in RIBI adjuvant induces a predominant type 1 immune response in vivo, characterized by relatively high levels of IFN-gamma and IL-2 and low levels of IL-4 and IL-10 mRNA in the draining prescapular lymph node. Ex vivo restimulation of draining lymph node lymphocytes with specific antigen resulted in proliferation and enhanced expression of IL-2 and IFN-gamma, whereas IL-4 and IL-10 transcript levels remained relatively low. These findings show that our previously described cytokine profiles of antigen-specific cloned T cell lines are representative of autologous in vivo responses and confirm that type 1 recall responses to B. bigemina RAP-1 can be evoked in immunized animals by native parasite antigen.

Ostertagia ostertagi: isolation and partial characterization of somatic and metabolic antigens.

One metabolic (ES) and two somatic extracts (AS and MS) were prepared from Ostertagia ostertagi. Partial characterization of the three preparations was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. In immunoblots, AS showed a low number of bands that reacted weakly with sera collected from infected calves. MS reacted strongly with the homologous sera, and a sharp group of bands appeared from 12 to 14 kDa, increasing in intensity as the infection progressed. ES showed a group of strongly immunogenic bands in the range of 16-22 kDa. The three preparations were also tested for reactivity with specific anti-Ostertagia antibodies in an enzyme-linked immunosorbant assay (ELISA). The ELISA results showed that all three worm extracts contained antigen epitopes recognized by circulating antibody in sera taken from O. ostertagi-infected calves. The strongest response was seen when antibodies of the IgG1 isotype were reacted with MS and ES. When sera taken from O. radiatum-infected calves were used both somatic extracts showed high levels of cross-reactivity.

Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses.

Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10(4)-10(5) N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti-Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora-specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.

Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns.

Twenty-six experimentally infected calves were monitored daily for oocyst excretion. All began excreting oocysts 3-6 days p.i. Most calves (n = 23) excreted oocysts for 6-9 days, with a daily range from 4 x 10(2) to 4.15 x 10(7) oocysts g(-1) of faeces. Over half the calves excreted peak numbers of oocysts 6-8 days p.i. Diarrhoea, observed intermittently beginning as early as day 3 p.i., lasted 4-16 days and varied greatly in severity from calf to calf. In a second study, nine of 18 calves were orally inoculated with 5 x 10(6) oocysts between birth and 2 days of age and nine remained uninfected. Monoclonal antibodies for cell surface markers indicated substantial increases in CD4+ and CD8+ T cells in the intraepithelial lymphocyte population of the ilea of infected calves at 7-9 days of age. RT-PCR demonstrated increases in mRNA for interleukin-12 and interferon-gamma that correlated with increases in both CD4+ and CD8 + intraepithelial lymphocyte cells. Increased mRNA for interleukin-12 and interferon-gamma from lamina propria lymphocytes correlated with increased numbers of CD8+ cells. No changes were found in interleukin-2, interleukin-4 or interleukin-10 mRNA levels. However, interleukin-15 mRNA, possibly from epithelial cells contaminating intraepithelial lymphocytes, was decreased in infected calves and had a negative correlation with increases in CD4+ and CD8+ cells. No differences were detected in mRNA levels for cytokines from lymph node lymphocytes.

The effect of experimental trichostrongyle infections of housed young calves on the subsequent course of natural infection on pasture.

The present studies were designed to investigate whether experimental, mixed trichostrongyle infections of stabled calves prior to their first grazing season could confer sufficient immunity to significantly reduce egg excretion after turnout, and thereby prevent loss-producing infections later on. The study comprised four groups, each of seven calves. During spring, two of the groups received two different dose levels of infective larvae twice weekly, and one group received larger larval doses at monthly intervals. One group served as non-experimentally exposed controls. In May all groups grazed separately on similarly contaminated plots. All experimental groups had reduced egg counts, and herbage infectivity of their plots was significantly lower than that of the controls. These findings were reflected in serum pepsinogen levels and in specific serum antibody responses. Additionally, clinical disease was only observed in the control group animals.

Effects of ionizing radiation on in vitro proliferative responses of porcine peripheral blood lymphocytes.

The radiosensitivity of in vitro proliferative responses of porcine peripheral blood lymphocytes (PBL) was assessed. PBL were stimulated by Con-A, PHA, culture supernates from mitogen-stimulated porcine lymphocytes, or in the case of antigen-primed swine, specific antigens. The resulting levels of proliferation were assessed by a determination of the level of incorporation of tritiated thymidine in vitro, and in some cases by the presence of blast cells in the cultures. Porcine PBL were found to be more radioresistant than either mouse PBL or mouse spleen cells. Irradiation levels of greater than 3000 rads were necessary to arrest Con-A or PHA-induced proliferative responses. Proliferation induced by lymphokines in the form of supernates from mitogen-stimulated lymphocyte cultures was arrested in PBL that had received 3000 rads prior to culture. Antigen-induced proliferative responses in primed porcine PBL populations were the most radiosensitive, in that a previous irradiation with 500 rads was sufficient to completely abolish a secondary in vitro proliferative response.

Isolation and phenotypic characterization of abomasal mucosal lymphocytes in the course of a primary Ostertagia ostertagi infection in calves.

Isolation and characterization of surface marker phenotypes of abomasal intraepithelial (IEL), lamina propria (LPL) and abomasal lymph node lymphocytes (ABLN) from uninfected calves were conducted, and the dynamics of change in these populations during the course of a primary Ostertagia ostertagi infection were defined. To obtain viable IEL and LPL from the abomasal mucosa of cattle, a modified isolation method was developed. The phenotypic characterization of abomasal lymphocytes was accomplished by indirect immunofluorescence staining. In uninfected animals, numbers of T cells exceeded the number of immunoglobulin-bearing cells in IEL, LPL and ABLN. The predominant T cell type in IEL and LPL was CD8+ cells, while the CD4+ T cell predominated in ABLN. Levels of activated cells and T cell receptor-1 gamma delta T cells were higher in IEL and LPL compared to ABLN. Within 3 weeks of infection, the number of lymphocytes recovered from the abomasal lamina propira and the mass of the ABLN was dramatically increased when compared to uninfected animals. Laser flow cytometric analysis demonstrated increased levels of immunoglobulin-bearing cells, gamma delta T cells, and activated T cells in IEL, LPL and ABLN in the infected animals. The greatest changes in LPL and ABLN took place during the first days of infection, and these changes were apparent throughout the 28 days covered by the experiment.

Cytokine profile induced by a primary infection with Ostertagia ostertagi in cattle.

Changes that occur in the local draining lymph nodes including, changes in cell surface markers and cytokine gene expression were studied over the first 4 weeks of a primary, Ostertagia ostertagi infection of the abomasum. Cells recovered from the abomasal lymph nodes (ABLN) after infection showed a decrease in the percentage of CD3+ cells, and an increase in the percentage of IgM+ cells and cells bearing the TcR1 marker. These changes were coincident with an increase in the proportion of activated cells (II-2R). Analysis of mitogen-stimulated ABLN cells by RNase protection assay (RPA) showed a dramatic reduction in IL-2 and IFN-gamma transcription after infection. In addition, analysis of unstimulated ABLN cells by competitive RT-PCR showed a similar decrease in demonstrable levels of IL-2 mRNA, but IL-10, IL-4 and IFN-gamma mRNA levels were elevated.

Porcine interleukin 2: parameters of production and biochemical characterization.

Interleukin 2 (IL2) or T cell growth factor (TCGF) has been characterized in a number of species but not in porcines. Porcine IL2 was detected in supernates (SN) of cultures of pig lymphocytes by: 1) the stimulation of the IL2-sensitive murine T cell line, CT6; 2) a costimulator assay involving porcine thymocytes; and 3) by the in vitro maintenance of antigen or mitogen-induced porcine lymphoblastoid cells. Porcine IL2 production by pig lymphocytes was induced by the mitogens Concanavalin A (Con A) Phytohemagglutiniin (PHA), and Pokeweed mitogen (PWM), but not by lipopolysaccharide (LPS). IL2 activity was demonstrated in the SN of mitogen-stimulated lymphocyte cultures as early as 24 hr after initiation of culture, reached peak levels at 48 hr, and decreased by 72 hr. Mitogens induced IL2 secretion by pig peripheral blood mononuclear cells, lymph node cells, and spleen cells, but not thymus cells. The cells responsible for IL2 production are presumptive T cells because: 1) they are nylon wool non-adherent; and 2) are non-surface-Ig bearing. In contrast, SN from cultures of surface Ig-positive cells had minimal IL2 activity. Porcine IL2 resembles rat and human IL2 in that it has an apparent molecular weight of approximately 15,000, and does not bind to DEAE-cellulose (DE-52) ion exchange columns equilibrated in 0.05 M sodium phosphate buffer (pH 7.6).

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus.

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.

Limited effect of recombinant porcine interleukin-12 on porcine lymphocytes due to a low level of IL-12 beta2 receptor.

The cytokine interleukin-12 (IL-12) is a key molecule in the regulation of CD4 + T cell development and specifically potentiates T helper 1 responses in mouse and man. However, biological effects mediated by IL-12 have not been well defined in pigs. Herein, recombinant porcine IL-12 (rPoIL-12) was expressed in a swine poxvirus system as a biologically active heterodimer and used to stimulate bovine or swine lymphoblast cells. After 3 days of incubation, only bovine blasts were responsive to the rPoIL-12 treatment as monitored by cell proliferation in several independent trials. Similarly, i.m. administration of rPoIL-12 in the hind leg of 3-week-old pigs indicated a reduction in the number of interferon-gamma (IFN-gamma) producing lymphocytes isolated from inguinal lymph nodes. The porcine IL-12R beta2 (IL-12Rbeta2) sequence was cloned and results generated by reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that the expression of IL-12R on porcine blasts as measured by the relative levels of IL-12Rbeta2 mRNA was less than that in bovine blasts and are in agreement with the reduced proliferation response of swine blast cells to rPoIL-12 treatment. Real time PCR analysis demonstrated that after PBMC stimulation, bovine blasts had an 11-fold increase in IL-12Rbeta2 mRNA levels while porcine blasts had almost no change. These data support a mechanism for IL-12 stimulation in swine inconsistent with that observed in conventional models.

Ostertagia ostertagi: changes in lymphoid populations in the local lymphoid tissues after primary or secondary infection.

The purpose of this study was to begin to define the changes in the local lymphoid tissues that accompany Ostertagia ostertagi infection in naive and immunized calves. Abomasal lymph nodes were taken from calves beginning as early as 2 days post-infection. Phenotypic changes in the resulting lymphocyte populations were assessed by flow cytometry utilizing monoclonal antibodies specific for the cell surface determinates CD2, CD4, and CD8. Changes in antigen specificity were determined by limiting dilution analysis utilizing antigen derived from fourth-stage O. ostertagi. Primary infection of naive calves caused a rapid 30-40% decrease in the percentage of T cells in the abomasal lymph nodes. This decrease in T cell percentage was due to a decrease in cells bearing the CD4 marker, a marker usually associated with helper T cells. Immunized calves were able to maintain normal T cell percentages of 50-60% for the first 5 weeks of infection. Immunization greatly increased the total number of Ostertagia-specific T cells in the abomasal lymph nodes owing to a marked increase in the size of the lymph nodes. Challenge infection of naive and immunized calves caused an increase in the frequency of parasite-specific T cells in both groups, but the increase was more rapid in the previously immunized calves. Within 5 weeks of infection, Ostertagia-specific cells could not be detected in the abomasal lymph nodes. These results indicate that the critical time period for expansion and regulation of Ostertagia-specific T cells in infected calves is early in the infection at a time that coincides with larval development. In addition, previous exposure to parasite antigens appears to result in more rapid responses and in the maintenance of normal ratios of T cell subpopulations in the draining lymphoid tissues.

Effects of gastrointestinal nematode infection on the ruminant immune system.

Gastrointestinal (GI) nematodes of ruminants evoke a wide variety of immune responses in their hosts. In terms of specific immune responses directed against parasite antigens, the resulting immune responses may vary from those that give strong protection from reinfection after a relatively light exposure (e.g. Oesophagostomum radiatum) to responses that are very weak and delayed in their onset (e.g. Ostertagia ostertagi). The nature of these protective immune responses has been covered in another section of the workshop and the purpose of this section will be to explore the nature of changes that occur in the immune system of infected animals and to discuss the effect of GI nematode infections upon the overall immunoresponsiveness of the host. The discussion will focus primarily on Ostertagia ostertagi because this parasite has received the most attention in published studies. The interaction of Ostertagia and the host immune system presents what appears to be an interesting contradiction. Protective immunity directed against the parasite is slow to arise and when compared to some of the other GI nematodes, is relatively weak. Although responses that reduce egg output in the feces or increase the number of larvae undergoing inhibition may occur after a relatively brief exposure (3-4 months), immune responses which reduce the number of parasites that can establish in the host are not evident until the animal's second year. Additionally, even older animals that have spent several seasons on infected pastures will have low numbers of Ostertagia in their abomasa, indicating that sterilizing immune responses against the parasite are uncommon. In spite of this apparent lack of specific protective immune responses, infections with Ostertagia induce profound changes in the host immune system. These changes include a tremendous expansion of both the number of lymphocytes in the local lymph nodes and the number of lymphoid cells in the mucosa of the abomasum. This expansion in cell numbers involves a shift away from a predominant classic T cell population (CD2 and CD3 positive), to a population where T cell percentages are decreased and B cells (immunoglobulin-bearing) and gamma-delta cells are increased. At the same time the expression of messenger RNAs for T cell cytokines (IL2, IL4, IL10 and gamma-interferon) is changed to that of increased expression of IL4 and IL10 and decreased expression of IL2 and perhaps of gamma-interferon. The reasons for these changes remain to be elucidated, but it is evident that the lack of protective immune responses is not the result of a poor exposure of the host to parasite products, or to the stomach being an immunoprivileged site. In fact, a superficial look at the responses elicited indicates that Ostertagia induces responses (the so-called TH2 mediated responses) that are widely considered to be the type of responses necessary for protection against GI nematodes. There are many factors that could lead to this apparent lack of immunity in the face of a strong stimulation of immune responses including: (1) the elicitation of suboptimal responses; (2) the failure of the abomasum to function as an efficient effector organ; (3) active evasion of the functional immune response by the parasite; and (4) that these classic responses are not protective in this particular ruminant-parasite system and that novel protective mechanisms may be required. The strong stimulation of the host gut immune system by Ostertagia and perhaps by other GI nematode infections, raises questions about the potential effects of such infections on the overall well-being of the host. A number of authors have indicated that Ostertagia infections may diminish the host's ability to mount subsequent immune responses to antigenic challenges such as vaccination against other infectious organisms. In addition, recent studies have indicated that infections with GI nematodes may result in increased circulatory levels of stress-related hormo

Limiting dilution analyses for the quantification of cellular immune responses in bovine ostertagiasis.

A sensitive limiting dilution analysis (LDA) was used to quantitate the local and systemic cellular immune response of cattle after immunization with keyhole limpet hemocyanin (KLH) and infection with Ostertagia ostertagi. The assay measures the proliferative response of bovine T-cells after in vitro stimulation with antigen. Interleukin 2 activity was supplied by supernates from mitogen-stimulated bovine peripheral blood lymphocytes (PBL) and accessory cell function was in the form of irradiated autologous PBL. The assay measures the response of a single cell and was most easily demonstrated in the lymph nodes draining the site of antigen inoculation. Comparison of cell frequencies and maximal responses generated in conventional proliferative assays showed several differences between the two assays. First, after antigen injection, the highest cell frequencies were seen in the draining lymph nodes within 3 days, and decreased by 10 days post-immunization. In contrast, in mass cultures maximal stimulation was not seen until 7-10 days after injection, but remained high up to 4 weeks after immunization. Second, at 17 days post-infection, a time of eruption of the parasite from the gastric glands, high frequencies of inducible cells were demonstrated by LDA in all lymphoid populations tested. In contrast, low levels of proliferation were seen in mass cultures. The reasons for these differences may include different sensitivities to suppression or more stringent requirements for specificity between the two assays. Finally, it was found that immunologically naive calves have relatively high frequencies of Ostertagia-specific cells in PBL, and that after infection these frequencies decrease. These results indicate either active suppression of the potential anti-Ostertagia response or an extra-vascularization of these cells to the site of infection.