Evidence for immunosuppression associated with Jembrana disease virus infection of cattle.

Jembrana disease virus (JDV) is a newly recognised bovine lentivirus causing an acute disease syndrome in Bali cattle (Bos javanicus) in Indonesia. We evaluated the effect of JDV infection on the antibody response to chicken ovalbumin (cOVA) and Brucella abortus Strain 19 in Bali cattle. In infected cattle the IgG and IgM response to cOVA was suppressed and delayed and the IgG response to B. abortus Strain 19 was delayed. The results indicate that the humoral immune response is suppressed and delayed in JDV infected cattle.     

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Bali cattle (Bos javanicus), which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5'- and 3'-long terminal repeats (LTRs), 0.4kb of truncated gag and 1.1kb of 3'-env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences, including gag, pol, vif, tat and rev, but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped, disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2)x10(6)CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as well. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus.     

Interleukin-6 expression in gut of parasite challenged sheep

Following challenge with Trichosirongylus colubrifonizis, increased numbers of T-cells and immunoglobulin responses are seen in the intestine of sheep immunised by repeated infection with live worms. IL-6 mRNA expression in the small intestine from T. colubriformis-immunised and naive sheep was determined by in situ hybridisation, whereas CD4(+), IgA(+), IgG(+) cells in the gut were evaluated by immunohistochemistry. There was constitutive expression of IL-6 mRNA by cells in the naive gut, and the number of these cells was increased by parasite challenge. There were corresponding increases in numbers of CD4(+) and TCR gamma/delta(+) T-cells and IgG(+) B-cells. Our data are consistent with a role for IL-6, perhaps produced by CD4(+) and/or TCR gamma/delta(+) T-cells or B-cells, in B-cell terminal differentiation. Infiltration of B-cells, particularly IgG(+) B-cells, may reflect parasite immunity in the host.     

Vascular lesions in lungs of Bali cattle with Jembrana disease.

Jembrana disease is an acute infectious disease of unknown etiology enzootic among Bali cattle (Bos javanicus) in Indonesia. Morphologic examination of 75 female Bali cattle between 18 months and 4 years old affected with Jembrana disease consistently revealed pulmonary granulomatous vascular lesions. The lesions were diffusely distributed throughout the lung. The principal lesion was the presence of a large number of intravascular macrophages that filled the lumina of pulmonary veins and pulmonary arteries of a vascular diameter of 20-200 microns, excluding the rest of blood cellular components. Concentric layers of perithelial cells also with plasma cells and macrophages were occasionally present around both veins and arteries. Infiltration of polymorphonuclear leukocytes or small lymphocytes was not seen. Destruction or necrosis of tissues or blood vessels was rarely seen. Because this vascular lesion was found in the lungs of all affected cattle examined, this change is useful for the postmortem diagnosis of Jembrana disease. Moreover, its presence could be used to distinguish Jembrana disease from malignant catarrhal fever and other lymphoreticular proliferative conditions that are frequently found among cattle in Indonesia.     

Characterization of age-related changes in bovine CD8+ T-cells

Evaluation of the changes induced by immunological interventions requires a baseline against which to compare those changes. The age-related changes in the CD8(+) T-cell population of cattle were studied. The results indicate that CD8(+) T-cells could be divided into γ/δ TCR1(+) and γ/δ TCR1(-) according to their expression of the γ/δ T-cell receptor. As a proportion, the CD8(+) γ/δ TCR1(+) population appears to increase with age. Within the CD8(+)γ/δ TCR1(-) a population of cells expressing a profile of surface molecules previously associated with effector memory T cells (CD45RO(+), CD62L(-), CD27(-), CD45RA(-) and CD28(-)) increases with age. Furthermore, a parallel increase with age in the proportion of CD8(+)CD45RO(+) T cells that express the cytotoxic granule protein perforin was observed. In peripheral tissues, namely lungs, it was found that the majority of CD8(+) T cells present expressed a phenotype indicative of previously primed T cells (high expression of CD45RO and perforin). In contrast, only a small population of memory CD8(+) T cells was present in lymphoid tissue where most of the CD8(+) T cells expressed a na?ve phenotype. In conclusion, in cattle, like in human, CD8(+) T cells that express a phenotype associated with antigen experience accumulate with age that may play a role in immunocompetence as the individual ages.     

Evidence for the presence of two bovine lentiviruses in the cattle population of Bali

Antibodies directed against two bovine lentiviruses, Jembrana disease virus (JDV) and bovine immunodeficiency virus (BIV), were detected in Balinese cattle sera using two new enzyme-linked immunosorbent assays (ELISAs) based on the combination of capsid (CA) protein and transmembrane (TM) peptides derived from JDV or BIV sequences.

Twenty eight of the 77 sera tested on the JDV ELISA showed anti-JDV antibodies with an unequal distribution of seropositive animals throughout the different districts of Bali. Furthermore, when 17 of the JDV positive sera were tested on Western blot, using the same JDV CA antigen, only 13 were judged positive confirming that the ELISA was a more sensitive technique for the detection of seropositive animals. Finally, 9 of the 49 JDV seronegative animals showed anti-BIV antibodies when tested on BIV-specific ELISA.

These two ELISAs appeared to be highly sensitive for the detection of anti-JDV and anti-BIV antibodies. Moreover, for the first time, animals showing antibodies against BIV were identified on the main island of Bali and on the JDV-free Nusa Penida island.     

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days.Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.     

Evidence for a viral aetiology of Jembrana disease in Bali cattle.

Jembrana disease is an acute infectious disease of Bali cattle (Bos javanicus) in Indonesia. High titres of the infectious agent are present in plasma during the febrile phase of the disease. The size of the agent determined by membrane filtration was between 50 and 100 nm, indicating it is a virus and not a rickettsia as previously proposed. Spherical virus-like particles of 75 to 130 nm diameter with a smooth membrane and frequently with an eccentric nucleoid were detected by electron microscopy in plasma from infected animals. The virus replicated in mononuclear cell cultures of peripheral blood origin but not in other cell cultures. The virus and the associated disease have characteristics consistent with viruses in the family Retroviridae.     

Parturition invokes changes in peripheral blood mononuclear cell populations in Holstein dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.     

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.     

Lymphatic organ development in dogs: major lymphocyte subsets and activity

In the present study, we have characterized lymphocyte subsets and activity in peripheral blood, spleen, mesenteric and popliteal lymph nodes in pups from birth till the age of one month and compared the results with the situation in the group of three adult dogs. In neonatal pups, lower numbers of CD3(+) T-cells were detected in both the spleen and peripheral blood than in lymph nodes. In contrast to the other compartments, CD21(+) B-cells prevailed in the spleen, which resulted in low values (<1) of the CD3(+)/CD21(+) ratio. Low numbers of CD8(+) lymphocytes were characteristic in all compartments immediately after birth; consequently a high CD4(+)/CD8(+) ratio has been calculated. Postnatal development was characterized by an increasing frequency of CD8(+) lymphocytes in all organs studied. Another typical feature of the early period of life was a relative decrease of B-cell numbers, which was compensated by an increasing proportion of T-lymphocytes, particularly in the peripheral blood and spleen. DNA synthesis in newborn pups' cells as measured by in vitro thymidine incorporation was surprisingly high in non-stimulated control samples, notably in the spleen. Further development of lymphocyte activity was characterized by the decline in spontaneous activity in all organs. Stimulation indices upon mitogen-induced proliferation increased proportionally to the decrease in spontaneous activity. Based on our experimental data, we have concluded that pups are born with a relatively competent immune system the structure of which, however, markedly develops during a few postnatal weeks.     

Distribution and activation of T-lymphocyte subsets in tuberculous bovine lymph-node granulomas

The immune response against mycobacterial infections is dependant upon a complex interaction between T lymphocytes and macrophages in the context of the granuloma. For this study, we performed the analysis of 18 stage I or II, and 13 stage III or IV granulomas found in lymph nodes from 8 experimentally and 2 naturally infected cattle. T-cell subpopulations (CD3(+), CD4(+), CD8(+), WC1(+), CD25(+)) were investigated by immunohistochemistry. In the majority of stage I/II lesions, CD8(+) and CD25(+) cells were predominantly found in the lymphocytic outer region of the granuloma, suggesting a possible role for activated CD8(+) cells in the initial attempt to restrain the granuloma growth. CD4(+) T cells appeared equally distributed in the lymphocytic mantle and in the internal areas of the granulomas. WC1(+) cells appeared interspersed among the macrophages. We speculated that this could indicate a role for these 2 subsets in the maintenance and the maturation of the granuloma. In stage III/IV lesions, all of the T-cell subsets investigated appeared interspersed among the mononuclear component of the granulomas. In general terms, there was a higher density of CD8(+) cells compared with CD4(+) cells. However, there was no sense of rimming effect for any of the investigated cell populations.     

Canine CD4(+)CD8(+) double positive T cells in peripheral blood have features of activated T cells

In dogs a CD4(+)CD8(+) double positive T cell subpopulation exists that has not been phenotypically defined yet. We demonstrate that canine CD4(+)CD8(+) T cells are mature CD1a(-) and TCRαβ(+) T cells. To analyse the activation potential of CD4(+)CD8(+) T cells, PBMC from dogs vaccinated against canine distemper virus (CDV) were re-stimulated with CDV. Upon antigen-specific stimulation, the CD4(+)CD8(+) T cell fraction increases and consists nearly exclusively of proliferated cells. Similarly, other features of activated effector/memory T cells such as up-regulation of CD25 and MHC-II as well as down-regulation of CD62L (L-selectin) were observed in CD4(+)CD8(+) T cells after stimulation. Canine CD4(+)CD8(+) T cells are less abundant, but more heterogeneous than porcine ones, comprising a small proportion expressing the β chain of CD8 in addition to the CD8α chain, like human CD4(+)CD8(+) T cells. In summary, this analysis provides the basis for functional characterisation of the in vivo relevance of CD4(+)CD8(+) T cells in T-cell mediated immunity.     

Ontogeny of systemic cellular immunity in the neonatal pig: correlation with the development of post-weaning multisystemic wasting syndrome

The aetiology of porcine post-weaning multisystemic wasting syndrome (PMWS) is poorly understood. Porcine circovirus type 2 (PCV-2) is an essential component of the experimental disease model for PMWS: however, evidence from experimental and field studies indicates that additional factors play a critical role in the aetiopathogenesis of PMWS. Current candidates include (1) immune stimulation (for example, via co-infection or vaccination), and (2) a novel infectious agent. A prospective, longitudinal case-control study was designed to investigate molecular triggers in leucocytes of neonatal piglets that may predispose to the development of PMWS. Blood samples were collected weekly from pigs (n=125) within five farms, from 1 week to 8 weeks of age: that is, before the appearance of clinical signs. Four colour flow cytometry was used to investigate changes in subsets of peripheral blood mononuclear cells, using monoclonal antibodies against the following cell associated markers; sIgG, CD3, MHCII dR, CD14, CD4a, CD8a, CD45RC, CD25, SWC3a, SWC8, CD163 and CD45. Sampling and laboratory analysis was supported by monitoring of clinical signs from 1 week to 20 weeks of age, or until disease supervened. At the conclusion of the study, 68 pigs (54%) were classified in Group 1 (no signs of clinical disease), 34 pigs (27%) in Group 2 (signs of clinical disease but not characteristic of PMWS), 17 pigs (14%) in Group 3 (suspect PMWS case) and 5 pigs (4%) in Group 4 (PMWS case). A single case of Porcine Dermatitis and Nephropathy syndrome (PDNS) was also diagnosed. Significant changes with age were demonstrated in clinically normal, neonatal pigs (Group 1), including an increase in B-cells and T-cells, and an increase in the proportion of total T-cells expressing MHCII. Within the T-cell subset, the proportion of CD8(+high) CD4(-) T-cells increased, in addition to the proportion of CD4(+) T-cells co-expressing CD8. Of the factors recorded, farm was found to have a highly significant effect on immune system development in the neonate. Comparison of Groups 1 and 4 cases identified significant differences between pigs which remained normal and those which subsequently developed PMWS. Pigs which went on to develop PMWS had a greater proportion of T-cells expressing MHCII in early life, higher mean intensity of expression of MHCII on T-cells, higher mean intensity of expression of MHCII on B cells and higher expression of CD25 on CD45RC(-) T-cells. These findings suggest that lymphocyte activation may be a key early event in the aetiology of PMWS.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Cattle infected with bovine leukaemia virus may not only develop persistent B-cell lymphocytosis but also persistent B-cell lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non-persistent lymphocytotic, PL-) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL- cattle and compared with six age-matched animals with persistent lymphocytosis (PL+) and five non-infected healthy controls (BLV-). In the PL- group, the percentage and number of surface immunoglobulin-positive (sIg+) B cells were significantly reduced. Whereas in BLV-cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg + and 24% were sIgM + B cells. In the PL- group, less than 20% of the PBL were sIg+ and sIgM+ B cells. Only 5% of the PBL co-expressed sIgM+ and CD5+ versus 16% in BLV-. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM + B cells co-expressing CD5 and CD11b; and (iii) equally both lambda- and K-type light chain B-cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5- and CD11b- sIgM+ B cells and CD2+ T cells did not differ. In PL+ animals, about 75% of the PBL were sIgM+ CD5+ B cells. These cells were of polyclonal origin, as light chains of the lambda- and K-type were expressed in a ratio of 4:1 (57.7% of PBL lambda+, 14% kappa+) as in BLV- animals (33.6% of PBL lambda+, 8.7% kappa+). In PL+ cattle the absolute number of B-cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T-cell numbers, the relative percentage of T-cells could be lower than in normal controls. The cause for the observed B cell decrease in PL- cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B-cell lymphopenia.     

CD4(+) T-cell responses against the VP1-unique region in individuals with recent and persistent parvovirus B19 infection

To date cellular immune responses against parvovirus B19 (B19) have not been studied extensively. The aim of this study was to examine the T-cell response against the VP1-unique region as the immunodominant part of the viral structural protein VP1 in individuals with different courses of B19 infection. Therefore, a group of 13 parvovirus-positive probands was separated into subgroups characterized for recent or acute, past or persistent infection by means of the presence of specific immunoglobulin (Ig)M and IgG isotypes and of viral DNA in blood and tissue. Transiently transfected B-cells expressing VP1-unique region were used in ELISpot assays to investigate T-cell responses directed against the VP1-unique region in peripheral blood mononuclear cells (PBMC) of individual donors. Significant numbers of interferon-gamma (IFN-gamma) secreting lymphocytes were detectable in PBMC of all individuals with recent, acute or persistent B19 infection, but not in PBMC of donors with past B19 infection and seronegative individuals. A more detailed analysis of IFN-gamma producing cells by intracellular cytokine staining by flow cytometry revealed, that CD4(+) T cells but not CD8(+) cytotoxic lymphocytes (CTL) were the major subpopulation of IFN-gamma producing cells. These data strongly suggest the need of virus protein production for the maintenance of VP1-unique region-specific CD4(+) T-helper cell responses in B19-infected individuals.