[Interleukin-2 for the treatment of cows with malignant catarrhal fever]. / Behandlung von Rindern mit Bösartigem Katarrhalfieber mit Interleukin-2.

The goal of this study was to investigate whether administration of interleukin-2 (IL-2) would improve the outcome of cows with malignant catarrhal fever (MCF). The study population consisted of ten healthy control cows and 22 cows with MCF. Nineteen cows with MCF and all of the controls were treated with either 2'500 U IL-2 or 25'000 U IL-2, administered intravenously. Three cows with MCF were not treated with IL-2 (MCF controls). All of the cows with MCF received danofloxacin, flunixin meglumine and intravenous fluid therapy. Blood samples for haematological and biochemical evaluation were collected once daily for six days in all cows. Of the 19 cows treated with IL-2, 13 were eutha nized because of deterioration. All cows with MCF that did not receive IL-2 died. The clinical condition of six cows treated with 2'500 U IL-2 gradually improved. Sur viving cows had significantly higher total leukocyte counts than cows that died or were euthanized. The main reason for leukopenia in non-surviving vs. surviv ing cows was persistent lymphopenia. Use of the lower IL-2 dose was associated with clinical recovery in some cows and this treatment might therefore be considered in valuable cows, provided that the lymphocyte count is within the reference interval.

Surgical stress influences cytokine content in autologous conditioned serum.

REASONS FOR PERFORMING STUDY: No recommendations have been made regarding the relative timing of blood collection for autologous conditioned serum (ACS) preparation and surgical procedures. OBJECTIVES: 1) To identify effects of surgical stress on cytokine levels in ACS, 2) identify haematological markers for prediction of cytokine production in ACS and 3) investigate the necessity for specialised ACS containers when preparing a cytokine-rich serum. STUDY DESIGN: Experimental in vitro study. METHODS: Blood was drawn from 15 stallions admitted for elective castration preoperatively and 22-24 h post operatively and incubated in ACS containers and plastic vacutainer tubes containing Z Serum Clot Activator. Concentrations of interleukin (IL)-1 receptor agonist (IL-1Ra), IL-10, IL-1ß, tumour necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß were determined in all serum samples and compared between preparation methods and sampling time by ANOVA. Changes in cytokine levels induced by incubation, defined as delta cytokine, were calculated by subtracting the baseline levels from the levels in incubated samples. Based on post operative serum amyloid A (SAA), horses were grouped into 'mild', moderate' and 'marked' surgical stress; delta cytokine levels in post operative samples were compared between these groups by ANOVA. RESULTS: Delta IGF-1 was significantly lower in post operative samples compared with preoperative. Horses in the 'marked' surgical stress group had significantly lower delta IL-1Ra and delta TGF-ß than the 'moderate' group and significantly lower delta IGF-1 than the 'mild' group. No association between cytokine levels and haematology variables were identified. Cytokine levels were comparable between serum prepared in blood tubes and in specialised ACS containers. CONCLUSIONS: Surgical stress influences the cytokine content in ACS. Useful predictors of cytokine production in ACS were not identified. Specialised ACS containers may not be necessary for preparation of a cytokine-rich serum.

Intestinal strictures, fibrous adhesions and high local interleukin-10 levels in goats infected naturally with Mycobacterium avium subsp. paratuberculosis.

This study describes pathological findings and their association with the production of interferon (IFN)-γ and interleukin (IL)-10 in goats infected naturally with Mycobacterium avium subsp. paratuberculosis (MAP). Twenty-seven goats were subjected to pathological examination. More than half of the animals had severe, diffuse, transmural granulomatous enteritis, often with abundant acid-fast bacilli (AFB), which was most evident in the proximal jejunum. Jejunal strictures and fibrous, peritoneal adhesions were findings that are not often reported in animals with paratuberculosis. Immunohistochemical labelling of IL-10 was seen within diffuse, granulomatous lesions and this may have prevented optimal local IFN-γ production and exacerbated the disease. However, since IFN-γ production was detected in cells from blood, jejunum and jejunal lymph nodes of goats with severe lesions by enzyme-linked immunosorbent assay, intracellular labelling and in-situ hybridization, the up-regulation of IL-10 might have been a consequence rather than a cause of the severe disease. The IL-10 labelling was co-localized with major histocompatibility complex (MHC) class II(+) cells, but rarely with CD4(+) cells. Comparable numbers of CD4(+) and CD8(+) T cells were recruited to both severe, diffuse lesions and small to moderate granulomatous lesions, while few T cells expressing the γδ form of the T-cell receptor were associated with both types of lesions.

Characterization of the immune response in the placenta of cattle experimentally infected with Neospora caninum in early gestation.

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days' gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (gammadelta) T-cell receptors (TCR), CD79alpha cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-gamma (IFN-gamma) were labelled by in-situ hybridization. Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3(+) lymphocytes, dominated by CD4(+) and gammadelta TCR(+) cells, with CD8(+) cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4(+) cells and NKp46(+) cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of gammadelta TCR(+) cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-gamma were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-gamma, suggesting that the parasite had not reached the placenta. The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno-fetal interface, resulting in infiltrations of CD4T cells, gammadelta T cells and NK cells and the subsequent production of IFN-gamma. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis.

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.

Characterization of macrophages and occurrence of T cells in intestinal lesions of subclinical paratuberculosis in goats.

The granulomatous lesions of subclinical paratuberculosis of goats were examined with emphasis on phenotypic characteristics of macrophages and the presence of different subpopulations of T cells. The macrophages in the granulomatous lesions were morphologically homogeneous in histological sections but showed varying expression of the macrophage marker CD68 (a glycoprotein found mainly in late endosomal and lysosomal membranes) and varying acid phosphatase activity. The lesional macrophages showed decreased expression of complement receptor 3 and major histocompatibility complex proteins, which are markers associated with phagocytosis and antigen-presentation, respectively. The granulomas showed low proliferation activity as measured by the proliferation-associated protein Ki-67, indicating that most cells were recruited to the lesions. Few apoptotic cells were demonstrated by the TUNEL technique, suggesting a low cell turnover in the lesions. CD4(+) T cells constituted the main T-cell population among the CD68(+) macrophages in the granulomatous lesions, and few CD8(+) T cells and gamma delta T cells were observed within the lesions, suggesting the limited ability of these cells to influence the granulomatous lesions in caprine subclinical paratuberculosis. Both WC1(+) and WC1(-) gamma delta T cells were present in the small intestinal wall, but the latter were the more numerous. No difference in the numbers of these cells was observed between the subclinically infected animals and control animals.

Modulation of leukocyte populations and immune responses in sheep experimentally infected with Anaplasma (formerly Ehrlichia) phagocytophilum.

Anaplasma phagocytophilum infection in sheep is characterized by an immune suppression as indicated by impaired antibody response, reduced lymphocyte response and reduced oxidative burst. The effect of A. phagocytophilum infection on leucocyte populations, especially lymphocytes, was therefore investigated in six sheep experimentally infected with A. phagocytophilum, and compared with leucocyte populations from control animals.To investigate the ability of the infection to interfere with the cellular and humoral responses to specific antigens, the animals were vaccinated with commercial vaccines at the time of experimental infection, and monitored for 56 days. There were reduced percentages of gammadelta T-cells and CD4+ T-cells in peripheral blood of infected animals throughout the study period, and these cell populations showed a down-regulation of CD25 expression; while there was a relative increase in CD8+ T-cells. The reduction in CD25+ gammadelta T-cells involved a subpopulation of WC1+ gammadelta T-cells. During the first 2 weeks of the study there were reduced percentages of B-cells and leukocytes expressing MHC II and CD11b, though this decrease changed to a relative increase later in the study. The relative reductions in leucocyte populations corresponded with the observed leucopenia during the first 3 weeks post-infection, which involved lymphocyte, neutrophil and eosinophil subsets [Vet. Immunol. Immunopathol. 86 (2002) 183]. There was a reduced expression of CD11b and CD14 on granulocytes during the first 2 weeks of the study, which corresponded with the previously reported leucopenia involving neutrophils and eosinophils. Antibody responses to vaccines, lymphocyte in vitro proliferative responses to antigens and mitogens, and in vitro IFN-gamma responses to antigens were reduced up to 4 weeks after infection.

DNA injection in combination with electroporation: a novel method for vaccination of farmed ruminants.

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635-40; Zucchelli et al. J Virol 2000;74:11598-607; Kadowaki et al. Vaccine 2000;18:2779-88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long-term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA-injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T-cell responses following electroporation were observed in hsp65 DNA-vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.

In vitro responses to purified protein derivate of caprine T lymphocytes following vaccination with live strains of Mycobacterium avium subsp paratuberculosis.

Live attenuated vaccines provide protection against intestinal lesions in goats infected with Mycobacterium avium subsp. paratuberculosis. To examine the role of different T lymphocyte subsets in the development of this protective immunity, CD4(+), CD8(+) and gamma delta T cell receptor (TCR)(+) cells from peripheral blood of goat kids vaccinated with live attenuated strains of M. a. paratuberculosis were studied. After in vitro stimulation with purified protein derivate, the expression of gamma-interferon (IFN-gamma) and the activation marker interleukin-2 receptor (IL-2R) was analysed by flow cytometry. A depletion experiment was performed, where the phenotypes and IL-2R expression was studied after stimulation of cultures depleted of a T lymphocyte subpopulation. Close to all of the IFN-gamma producing cells were of the CD4(+) subset, while only a small number were CD8(+) cells. The gamma delta TCR(+) cells were highly activated, but did not produce IFN-gamma after in vitro stimulation. Depletion of CD4(+) cells lead to a decrease in the percentage of total gamma delta TCR(+) cells and gamma delta TCR(+)IL2-R(+) cells. Removing the gamma delta TCR(+) cells increased the relative numbers of CD4(+), but not the CD4(+)IL-2R(+) cells. Insight into the in vitro recall responses of T cell subsets from animals vaccinated with live paratuberculosis vaccines is essential in the development of more efficient vaccines.

Lesions in subclinical paratuberculosis of goats are associated with persistent gut-associated lymphoid tissue.

The organized gut-associated lymphoid tissue (the Peyer's patches [PPs] of domestic ruminants) is an important site of lesions caused by Mycobacterium avium subsp. paratuberculosis. To investigate the association between PP morphology and the lesions of paratuberculosis in goats, two experiments were performed. Five healthy kids aged 4-5 weeks were examined and the morphology of organized lymphoid tissue in the small intestine was described. Morphological similarities were observed between the ileocaecal-valve PP (ICVPP) and the jejunal PPs (JPPs), with pear-shaped follicles, large submucosal interfollicular T-cell areas, and many intraepithelial leucocytes in the follicle-associated epithelium. The ileal PP (IPP) consisted of elongated follicles, small T-cell areas and few intraepithelial leucocytes. The association between these three locations of PPs and lesions of paratuberculosis was then studied in seven goats inoculated with M. a. paratuberculosis at 5-8 weeks of age and killed 2 years later, while in the subclinical phase of infection. Gross lesions were recorded in five animals and microscopic lesions were observed in the intestine and mesenteric lymph nodes of six animals. The lesions in the small intestine were mainly located in the PPs of the mid-jejunum (JPPs) and ICVPP. Lesions were not present in the intestinal segments that had contained IPP, which had undergone involution during the first 12-18 months of life. These observations indicate that the persistent organized lymphoid tissue in the JPPs and ICVPP, but not the involuted IPP, sustains the development of granulomatous inflammation due to paratuberculosis during the subclinical phase of infection.

Localisation of CD25+ cells and MHCII+ cells in lymph nodes draining Mycobacterium avium subsp. paratuberculosis vaccination granuloma and the presence of a systemic immune response.

Vaccination of goat kids against paratuberculosis protects against lesions and clinical disease. The systemic cellular response was studied in goat kids 3-9 weeks after vaccination. Peripheral blood cells showed increased interferon-gamma production and expression of interleukin-2 receptor (CD25) after stimulation with Mycobacterium avium subsp. paratuberculosis antigens. The lymph node draining the vaccination granuloma was studied three weeks after vaccination in a parallel group of goat kids. In deep cortex, MHCII+ cells were observed surrounded by CD4+ T-cells, while follicular hypertrophy and hyperplasia were prominent in the subcapsular region and along connective tissue trabecula. Comparison of the local and systemic immune responses revealed an inverse relationship between CD25+ T-cells in the lymph node deep cortex and cells in peripheral blood that up-regulate CD25 upon in vitro stimulation, suggesting that activated and regulatory T-cells in the local lymph node influence the level of circulating antigen-specific T-cells following vaccination against paratuberculosis in goats.

Functions of neutrophils in sheep experimentally infected with Ehrlichia phagocytophila.

Ehrlichia phagocytophila infection in sheep is characterized by persistent neutropaenia, indicative of decreased phagocytic capacity. This predisposes infected animals to other infections. A whole blood flow cytometrical method was used to document the degree and extent of reduced phagocytic and respiratory burst activity in phagocytes during an experimental infection with E. phagocytophila, and monitored until 56 days post-infection. Six sheep at 5 months of age were inoculated with an intravenous injection of infected blood. Six age-matched sheep were used as controls. A period of reduced respiratory burst lasting up to Day 17 post-infection was recorded. The population of cells showing phagocytic activity without respiratory burst was larger in the infected animals compared to controls up to Day 45 post-infection.

Reduced levels of total leukocytes and neutrophils in Norwegian cattle selected for decreased mastitis incidence.

Two groups of Norwegian Cattle showing significant genetic differences in clinical mastitis susceptibility were examined for hematological changes at three stages of lactation. Blood samples were taken from 91 healthy Norwegian Cattle cows and heifers belonging either to a high protein yield group or to a low clinical mastitis group and analyzed for hematological properties and serum cortisol levels at three stages of lactation. All animals were free of intramammary infections at the time of sampling. All cows had increased total white blood cells, neutrophils, and neutrophil/lymphocyte ratio as parturition approached, with peak levels at parturition. Cows selected for low clinical mastitis had lower levels of total white blood cells and neutrophils compared with cows selected for high protein yield throughout all three periods. The difference was significant prepartum. Cows from the low clinical mastitis group also had lower neutrophil/lymphocyte ratios and lower serum cortisol levels than those in the high protein yield group. A significant positive correlation was found between cortisol and total white blood cells and neutrophils, respectively. We conclude that selection for lower mastitis incidence in the low clinical mastitis group is associated with a significant decrease in total white blood cells and neutrophils in blood prepartum. Results from the present study, and the genetic difference in mastitis incidence observed in the groups, indicates that increased leukocyte mobilization at certain stages of lactation may be associated with increased susceptibility to mastitis.

Identification of residues within the extracellular domain 1 of bovine Fc gamma 2R essential for binding bovine IgG2.

Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFc gamma 2R initiates the killing process. bFc gamma 2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian Fc gamma Rs, bFc gamma 2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and Fc alpha RI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715-1722) that like these proteins (and unlike other Fc gamma Rs), bFc gamma 2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFc gamma 2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F-G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.

Innate IFN-gamma production in cattle in response to MPP14, a secreted protein from Mycobacterium avium subsp. Paratuberculosis.

Calves experimentally infected with Mycobacterium avium subsp. paratuberculosis and uninfected calves were tested for interferon(IFN)-gamma production after stimulation with purified protein derivative from M. avium subsp. paratuberculosis (PPDp) or a secreted 14 kDa protein (MPP14) specific for the M. avium-intracellulare-scrofulaceum (MAIS) complex. Several calves in both groups responded strongly up to about 5 months to both antigens. Two uninfected calves responded repeatedly, but not always, to MPP14 and PPDp throughout the study. The responses in the uninfected animals seemed to be independent of cell contact between the antigen presenting cells (APC) and the responding population. The supernatant from adherent cells stimulated with MPP14 induced similar levels of IFN-gamma production in CD14+/B-cell depleted peripheral blood mononuclear cells (PBMC) as when the antigen was used directly on PBMC. In contrast, APC/T-cell contact was necessary to induce the IFN-gamma production in infected animals, suggesting that both innate and adaptive IFN-gamma production in response to MPP14 could occur. CD8+ cells contributed to some of the IFN-gamma production in response to MPP14, but the rest could not be explained, while CD4+ cells were responsible for the adaptive response to PPDp. This study showed that secreted proteins could induce innate IFN-gamma production that interferes with diagnostic testing using the IFN-gamma-test.

Cloning and sequencing of a cDNA encoding the bovine FcR gamma chain.

Phagocytic cells of the immune system express specific receptors for the Fc region of immunoglobulins (FcRs). In humans, most FcRs for IgG (FcgammaR), IgA (FcalphaR) and IgE (FcvarepsilonR) consist of an immunoglobulin (Ig) -binding subunit associated with a specialized signaling molecule, the FcR gamma chain. The FcR gamma chain is crucial for the transmission of intracellular signals following receptor ligation. In cattle, however, although four distinct complimentary DNAs (cDNAs) encoding IgG-binding subunits have been described (corresponding to bovine FcgammaRI, FcgammaRII, FcgammaRIII, and Fcgamma2R), virtually, nothing is known about signal transduction via bovine FcRs. Therefore, in this study, a cDNA encoding the bovine FcR gamma chain was cloned. The cDNA is 258 base pairs long and encodes a protein of 85 amino-acids. The mature protein shows high homology with the FcR gamma chains from several other species. Interestingly, the cytoplasmic domain of the bovine FcR gamma chain is one amino-acid shorter than those previously described. Cloning of a cDNA encoding, the bovine FcR gamma chain will allow for a better understanding of signal transduction processes triggered by bovine FcRs.

Subclinical paratuberculosis in goats following experimental infection. An immunological and microbiological study.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

Kinetics of IL-2 receptor expression on lymphocyte subsets from goats infected with Mycobacterium avium subsp. paratuberculosis after specific in vitro stimulation.

Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.

The use of interleukin-2 receptor expression as a marker of cell-mediated immunity in goats experimentally infected with Mycobacterium avium ssp. paratuberculosis.

The purpose of the present work was to demonstrate cell-mediated immune response to paratuberculosis in experimentally infected animals, using quantification of interleukin-2 receptor (IL-2R) expression on activated lymphocytes by means of in vitro stimulation with Mycobacterium avium ssp. paratuberculosis-derived purified protein derivative (PPDp). A whole-blood technique was developed, and optimal conditions for quantification of IL-2R expression on caprine lymphocytes, using monoclonal antibodies (anti-bovine IL-2R-alpha) and low cytometrical analysis, were determined. Different PPDp-antigen concentrations and incubation times were compared. The whole-blood method was also compared to the more traditional IL-2R assay using peripheral blood mononuclear cultures (Hesketh et al., 1993). Cross-reactivity to Mycobacterium avium was studied at different mycobacteria-PPD concentrations. An immune response could be demonstrated in animals infected with Mycobacterium avium ssp. paratuberculosis. We found that a PPDp concentration of 10microgml(-1) together with an incubation time of 72h, gave the best results using the whole-blood method. The whole-blood method eliminates many laborious steps involved in lymphocyte separation, and the effects of all the constituents of blood are expressed in a way which corresponds more to in vivo conditions. The risk of selecting subpopulations of lymphocytes during cell separation is avoided.

Immunohistochemical identification of caprine arthritis-encephalitis virus in paraffin-embedded specimens from naturally infected goats.

The expression of caprine arthritis-encephalitis virus capsid protein was studied in seropositive naturally infected asymptomatic goals (10< seropositive naturally infected encephalitic kids (12) and goats (4), and noninfected control goats (3). Rabbit antiserum to recombinant viral capsid and matrix proteins were used in a biotin-streptavidin-alkaline phosphatase complex immunohistochemical method on sections of formalin- and ethanol-fixed tissue specimens. Macrophages in inflamed areas of the lung (8/12), in the brain (5/16), and in the spinal cord (4/16) from encephalitic animals harbored viral antigens, as revealed by immunohistochemistry and use of a capsid protein-specific antiserum. Altogether 12/16 encephalitic animals tested positive for viral antigen. Viral antigens were found in 5/10 seropositive asymptomatic goals in macrophages located in the lung (3), the udder (1), and the medulla of lymph nodes (4). None of the control animals tested positive for viral antigen. Ethanol fixation showed highest sensitivity, and the lowest antigen concentration that revealed a positive signal discernible from background was twofold higher in ethanol-fixed specimens than in formalin-fixed specimens. The evaluation was performed on artificial antigen substrates embedded with defined concentrations of recombinant viral capsid protein. Immunohistochemistry is a valuable supplement to the methods presently available for diagnosis in cases suspicious of caprine arthritis-encephalitis.