Polymorphic expression in the CD8alpha chain surface receptor of African lions (Panthera leo).

Free-ranging African lion (Panthera leo) peripheral blood mononuclear cells (PBMC) were examined using flow cytometry and antibodies developed for use in the domestic cat to determine if phenotypic changes occurred in lion lymphocytes as a result of feline immunodeficiency virus (FIV) infection. The percentage of CD8 cells from lion peripheral blood was considerably lower than in the domestic cat. Lions with elevated levels of CD8+ cells were typically infected with FIV, similar to observations in the domestic cat. Antibodies against the alpha chain of the CD8 receptor (monoclonal antibody (mAb) 3.357) did not react consistently in all lions examined. Flow cytometric analysis determined that approximately 82 and 80% of the animals from Kruger and Hluhluwe-Umfolozi National Parks in South Africa reacted with the monoclonal antibody against the alpha chain of CD8 receptor, while only 17% of the lions in Etosha National Park in Namibia cross-reacted with the CD8alpha chain. There was no apparent correlation between FIV status and CD8alpha chain reactivity. The relative isolation of Etosha from the other two parks could explain the marked difference in CD8alpha chain expression and suggests that lions similar to other mammalian species demonstrate polymorphic expression of the CD8alpha chain (197).     

An Immunohistochemical Study of Bovine Palatine and Pharyngeal Tonsils at 21, 60 and 300 Days of Age

An immunohistochemical study was performed on three groups of young cattle (21, 60 and 300 days of age). Tonsils (palatine and pharyngeal) and mucosae (nasal and oral) were removed. Eight monoclonal antibodies (specific for CD3, CD2, CD4, CD8, WC1, cell-surface IgM, cell-surface IgG and MHC class II molecules) and an avidin/biotin complex method on frozen sections were used. The immunological cytoarchitecture of bovine tonsils is similar to that of human tonsils. Nevertheless, these lymphoid tissues are not fully developed during the first weeks of life: T and B dependent areas not well-differentiated, few germinal centres, few intra-epithelial WC1 + T lymphocytes. In contrast, at 2 months, tonsils possess all the elements of a mucosa-associated lymphoid tissue (MALT). Tonsillar or mucosal epithelium is infiltrated by a large number of CD8+, WC1+ T lymphocytes and cells which express MHC class II molecules. Between 21 and 60 days, the number of WC1+ T lymphocytes increase markedly in the tonsillar epithelium. These results accredit the hypothesis that the presence of antigens has an effect on the localization of these lymphocytes at these sites.     

Toll-like receptor expression in feline lymphoid tissues

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.     

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.     

Characterization of a feline homologue of the alphaE integrin subunit (CD103) reveals high specificity for intra-epithelial lymphocytes

The characteristics of a feline homologue of the alphaE integrin (CD103), defined by two murine monoclonal antibodies, Fe7.1B8 (IgG1) and Fe7.2D8 (IgG1), are described. These antibodies recognized 75% of intra-epithelial (range 59-88%) and 40% of lamina proprial (range 28-46%) T cells of the intestinal mucosal tissue of the small intestine in contrast with approximately 2% of peripheral blood lymphocytes. Both antibodies immunoprecipitated a 180 kDa protein from biotinylated feline intra-epithelial mucosal leukocytes consistent with the alphaE integrin subunit in conjunction with a 120 kDa protein consistent with the beta7 subunit. The nucleotide sequence of feline alphaE integrin, generated from molecular cloning of the feline alphaE encoding cDNA, is also reported. This feline molecule shares 72% sequence homology with human and 69% homology with murine and rat counterparts. Homology includes the presence of an X (extra) domain, that appears unique to alphaE molecules as described for human, rat and mouse, as well as areas of homology common to other alpha integrins. Of note is a typical I (inserted) domain, the presence of seven repeat regions, and highly conserved sequences in the cytoplasmic tail. Transfection studies demonstrated that both antibodies recognized an extracellular component which encompassed the X and I domains of the cloned alphaE integrin subunit. These studies demonstrate that the pattern of tissue distribution, biochemical characteristics, and cDNA sequence of the feline alphaE integrin subunit are largely similar to that described for other species.     

Characterisation of immunosuppression in rabbits after infection with myxoma virus

Myxoma virus (MXV) causes the systemic disease myxomatosis in the European rabbit. Despite many in vitro studies on the function of MXV immunomodulatory proteins and detailed molecular knowledge of virus, little is known about the dynamics of interaction of the virus with the integrated host-immune system during infection. In this study changes in haematological profile, changes in lymphocyte subset distribution and non-specific proliferation activity of lymphocytes from different lymphoid compartments on the 2nd, 4th, 6th, 9th and 11th day after experimental infection of rabbits with MXV strain Lausanne was characterised. The relationship between alterations of immune parameters and dynamic of virus dissemination through the body was investigated. Haematological changes included moderate leucopenia with significant lymphopenia, neutrophilia, monocytosis and eosinopenia. A decrease of T cells including CD4+ and CD8+ and increase of CD79alpha+ were observed in draining popliteal lymph node 4 days after virus inoculation. From day 6, comparable changes were seen in collateral popliteal lymph node, spleen and peripheral blood. From day 9, the mentioned lymphocyte subsets tended to reach their original state in all of these lymphocyte compartments except draining popliteal lymph node. In thymus, MXV infection affected mainly CD4+CD8+ double positive thymocytes. On the other hand, proliferation activity of lymphocytes determined by the proliferation assay with plant-derived mitogens was significantly reduced from day 4 or 6 and remained reduced until the end of experiment in all observed lymphoid organs. Presence of MXV in respective lymphoid compartments preceded changes in lymphocyte subset distribution or lymphocyte activity.     

Lymphoid follicles in the gastric mucosa of dogs. Distribution and lymphocyte phenotypes

The occurrence and distribution of lymphoid follicles within the stomachs of 36 dogs that dis not have macroscopic gastric lesions are presented. The dogs ranged in age from less than 1 year to over 13 years. The number of follicles varied between the different regions of the stomach, being most numerous (15.6 follicles cm⁻²) and uniform in size (about 1 mm in diameter) in the fundus. The number and size of follicles in the antrum varied widely between dogs. Age-related changes in the distribution of follicles were not found following simple linear regression analysis. The phenotypes of lymphocytes in gastric lymphoid follicles of nine dogs aged from less than 1 year to 5 years were determined using monoclonal antibodies specific for canine leucocyte antigens and an indirect immunoperoxidase technique. The follicles had an organized distribution of lymphocytes subsets in that a predominantly B cell area contained some CD4+ cells and very few CD8+ cells and was adjacent to an area containing mostly T cells. Computer-assisted morphometric analysis was used to quantify the overall presence of the various lymphocyte subpopulations. Follicles in the fundus and body regions possessed similar percentages of lymphocytes averaging 42%, 22% and 3% of the area occupied by B cells, CD4+ and CD8+ cells, respectively. It is concluded that lymphoid follicles are a normal constituent of the canine gastric mucosa and possess a lymphocyte composition similar to that reported by others for solitary intestinal follicles.     

健康中国恒河猴各淋巴组织中T淋巴细胞表型及分布

试验旨在明确T淋巴细胞在中国恒河猴各组织中的表型与分布,为疾病模型研究提供基础数据。取外周血、腹股沟淋巴结、肠系膜淋巴结及肠道组织,从中分离出淋巴细胞。使用流式细胞术检测分析各种表型的淋巴细胞在组织间的分布。结果表明,淋巴结中CD4⁺ /CD8⁺ T细胞比值高于外周血,肠道固有层中最低,三者差异显著。记忆性T细胞在外周血和肠道固有层T细胞中比重较大,而淋巴结中主要为幼稚T细胞。CD4⁺ T细胞中中心记忆T细胞Tcm为主要亚群,而CD8⁺ T细胞主要为效应记忆细胞Tem。外周血与肠道固有层中增殖T细胞比例相当,而淋巴结中T细胞增殖水平相对较低。各组织中CXCR4受体表达量普遍高于CCR5受体,其中肠道固有层CCR5受体表达水平最高。值得注意的是,有一小群表型CD3⁺ CD4⁺ CD8low的细胞仅在肠道固有层中存在,经分析其功能活性应高于肠道CD4单阳性T细胞。因此,测定了健康中国恒河猴各表型T淋巴细胞在多种淋巴组织中的基础数值,为相关模型研究奠定基础。     

Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8(alpha)(alpha) phenotype

Clinicopathologic and immunophenotypic characteristics of large granular lymphocyte (LGL) neoplasia in 21 cats were examined. All cats were domestic short (19) or long hair (2) with a mean age of 9.3 years at diagnosis. Increased peripheral blood LGL counts were present in 18/21 cats. Neutrophilia (12/21 cats) and increased serum liver enzymes (7/12), total and direct bilirubin (7/13), BUN (5/14), and creatinine (2/14) were observed. Cats usually presented with advanced disease and none survived longer than 84 days (mean 18.8 days) postdiagnosis. Cytologically, LGLs had a mature (6/21), immature (13/21), or mixed (2/21) morphology. Necropsy lesions consisted of neoplastic lymphoid infiltrates in the jejunum, ileum, and duodenum in decreasing order of frequency. In the small intestine, mucosal ulceration (9/13) and epitheliotropism of neoplastic cells (9/13) were common. Neoplastic infiltrates were also present in the mesenteric lymph nodes (13/13), liver (12/13), spleen (8/13), kidneys (5/7), and bone marrow (5/7). A T cell phenotype (CD3epsilon+) characterized LGL neoplasia in 19/21 cases. A CD8alphaalpha+ cytotoxic/suppressor phenotype was present in 12/19 T cell tumors, 2 had a CD4+CD8alphaalpha phenotype, 3 had a CD4-CD8- phenotype, and 2 were CD4+ helper T cells. CD8beta chain expression was not detected in any instance. In two cats, a B or T cell origin could not be established. CD103 was expressed by 11 of 19 (58%) of the lymphomas tested. The immunophenotypic features shared by neoplastic LGLs in the cat and feline intestinal intraepithelial lymphocytes (IELs) support a small intestinal IEL origin for feline LGL lymphoma.     

A novel "small round virus" inducing poult enteritis and mortality syndrome and associated immune alterations

The role of a novel "small round virus" (SRV) isolated from poult enteritis and mortality syndrome (PEMS) cases in inducing PEMS and associated immune alterations was examined in this study. Specific-pathogen-free and conventional poults were orally challenged with SRV and/or turkey coronavirus and monitored for clinical signs. Intestines, thymus, bursa, and spleens were examined for SRV antigen at various days postinoculation (DPI). Peripheral blood lymphocytes (PBLs), thymocytes, and splenic lymphocytes from inoculated poults or lymphocytes isolated from healthy poults after incubation with SRV in vitro were examined for lymphoproliferative potential against concanavalin A (Con A). The incidence of lymphocyte subpopulations in the peripheral blood and thymic lymphocytes of SRV-challenged poults was examined by flow cytometry. The results of these studies showed that the SRV challenge induced diarrhea, growth suppression, and atrophy of thymus and bursa resembling those of PEMS in field and/or experimental infections. The SRV antigen was detected in intestinal tissues soon after infection (i.e., at 2 and 4 DPI), whereas lymphoid tissues such as thymus, bursa, and spleen were positive for SRV antigen starting at 4 DPI until 8 DPI, suggesting virus translocation to lymphoid organs. The responsiveness of PBLs to Con A at 2 DPI was significantly reduced in all virus challenge groups (e.g., 28% and 22% in the SRV-alone group in studies 1 and 2, respectively) below the uninfected group. However, this suppressed response was no longer evident in the SRV group by 7 DPI. The SRV incubation with normal thymocytes and splenocytes in vitro resulted in significantly reduced lymphoproliferative response against Con A (41.2% and 10.49% reductions at 1:50 SRV dilution vs. controls in thymocytes and splenocytes, respectively). Flow cytometry analysis revealed a sudden decline at 2 DPI in the numbers of CD4- CD8+ lymphocyte subset in PBLs of SRV-infected poults. However, by 8 DPI, SRV-challenged poults had relatively higher CD4- CD8+ lymphocytes in PBLs. On the contrary, thymocytes had higher percentages of CD4- CD8+ lymphocytes at 2 and 4 DPI and reached comparable levels at 8 DPI in controls and SRV-infected poults. No differences were observed in CD4+ CD8- lymphocyte numbers in controls vs. SRV-infected poults. The findings of these studies imply that SRV may be a promising primary etiologic agent of PEMS. Furthermore, the SRV infection may compromise the lymphocyte-mediated immune defenses by reducing lymphoproliferation and the CD4- CD8+ (presumably T-cytotoxic cells) lymphocytes during the acute stage of SRV infection.     

Dynamics and responsiveness of T-lymphocytes in secondary lymphoid organs of rabbits developing immunity to Eimeria intestinalis

Primary infection with Eimeria intestinalis confers very effective immunity against further infections in rabbits. This study was designed to determine the onset of the immune response in primary-infected rabbits and to characterise the immune status of protected rabbits. Variations in kinetics of CD4+ and CD8+ T-cell subpopulations were followed after primary infection at the intestinal sites of penetration (duodenum) and development (ileum), in mesenteric lymph nodes (MLN) and in the spleen. The response against the parasite was measured by specific lymphocyte proliferation in the spleen and MLN and by determining specific IgG titres in serum. The mucosal immune response was strong after primary infection and was characterised by (i) transient increase in the percentages of intestinal CD4+ lymphocytes and MLN CD8+ lymphocytes 14 days PI and (ii) strong increase in the percentages of intestinal CD8+ lymphocytes from 14 days PI persisting throughout further infections. Extensive infiltration of the lamina propria with CD8+ lymphocytes was observed 14 days PI. The specific proliferative response started between 7 and 14 days PI in MLN but remained undetectable in spleens for up to 21 days, in contrast to “immunised” rabbits. The fact that systemic immune responses were low after primary infection, in contrast to indicators of mucosal immune responsiveness, suggests that protection of rabbits against E. intestinalis infection is due to an effective mucosal immune response, and that systemic responses that increase after successive infections are only reflections of repeated encounters with parasite antigens.     

Cervical thymoma originating in ectopic thymic tissue in a cat

Abstract: An 11‐year‐old female spayed domestic shorthair cat was referred to The Ohio State University Veterinary Teaching Hospital (OSU‐VTH) for evaluation of a 6 × 4 × 3.5 cm mass in the left midcervical region causing increased respiratory sounds and lateral deviation of the trachea. A fine needle aspirate of the mass was obtained before referral and the cytology results were compatible with a reactive lymph node. Immunocytochemistry showed increased numbers of CD3+ T lymphocytes and small numbers of CD20+ and CD79a+ medium to large lymphocytes. Differential diagnoses from the referral pathologist were T‐cell‐rich B‐cell lymphoma and feline Hodgkin's‐like lymphoma. A subsequent fine needle aspirate performed at the OSU‐VTH showed similar results. On flow cytometry the majority of cells were CD3+ T lymphocytes that were double positive for CD4 and CD8 (73%), compatible with either a double‐positive (CD4+CD8+) T‐cell lymphoma or lymphocytes from ectopic thymic tissue. The mass was surgically removed. Histopathology and immunohistochemistry of the mass revealed a predominant population of CD3+ small lymphocytes and small numbers of medium to large lymphocytes with moderate anisocytosis and anysokaryosis. A population of cytokeratin‐positive epithelial cells surrounded small microcystic structures filled with eosinophilic material and structures interpreted as Hassall's corpuscles. These findings were consistent with thymic tissue and a diagnosis of ectopic thymoma was made. PCR results for lymphocyte antigen receptor rearrangement (PARR) were negative. The cat had no evidence of disease 16 months after removal of the mass. To our knowledge this is the first report of an ectopic cervical thymoma in a cat. The clinical and diagnostic features of this unusual case will be useful in helping veterinarians and pathologists obtain a presurgical diagnosis and establish a prognosis for similar lesions.     

A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes

Flow cytometry is becoming a commonly used technique to characterize a variety of cells. It provides a powerful application to rapidly determine the relative percentages of T-lymphocyte subsets and B-lymphocytes. The effectiveness of its application, however, is dependent on standardization, especially in a clinical setting. Application of flow cytometry to veterinary diagnostics has been limited by the unavailability of reagents and by the unstandardized characterization of normal values using antibodies not commercially available, but typically provided through the generosity of other researchers. This paper presents a standardized gating protocol, and average values and ranges observed for normal canine and feline blood lymphocytes using commercially available antibodies to cell surface markers for CD5, CD3, CD4, CD8, MHC II, and B lymphocytes. The averages for these markers on gated lymphocytes were as follows: Canine CD5 83.3%, Canine CD4 45.0%, Canine CD8 28.8%, Canine MHC II 98.0%, Canine B Cell 12.9%, Canine CD4/CD8 ratio 1.87, Feline T lymphocytes 77.3%, Feline CD4 44.5%, Feline CD8 25.7%, Feline B Cell 24.1%, Feline CD4/CD8 Ratio 1.75. Normal values were also established for a mixed breed group of dogs, and old versus young dogs. This information will provide researchers and clinicians with a standardized protocol for gating, which establishes a basis for comparison between techniques, and a measure of phenotypic percentages for flow cytometry in normal dogs and cats based on this standardization and commercially available antibodies.     

Early events in the immunopathogenesis of feline retrovirus infections.

Feline leukemia virus and feline immunodeficiency virus (FIV) are lymphotropic retroviruses that cause a wide range of diseases in domestic cats. Although it is known that both viruses are capable of infecting T lymphocytes and that infected cats are lymphopenic, it was not known how infection with either virus might alter specific lymphocyte subpopulations. Using a panel of monoclonal antibodies to feline lymphocyte subpopulations, we examined, by use of flow cytometric analysis, lymphocyte changes in cats naturally infected with FeLV or FIV and explored the early stages in the immunopathogenesis of experimentally induced infection with these viruses. Both groups of naturally infected cats had T-cell lymphopenia. In the FIV-infected cats, the T-cell decrease was principally attributable to loss of CD4+ cells, whereas CD8+ and B-cell numbers remained normal. This led to inversion of the CD4+ to CD8+ ratio in these cats. In contrast, the T-cell lymphopenia in FeLV-infected cats resulted from decrease in CD4+ and CD8+ cells, which led to a CD4+ to CD8+ ratio within normal limits. Experimentally induced infection with these 2 viruses supported these findings. Infection with FIV induced early (10 weeks after infection), chronic inversion of the CD4+ to CD8+ ratio. In contrast, infection with FeLV did not alter CD4+ to CD8+ ratio in the first 20 weeks after infection.     

禽网状内皮组织增生病病毒感染对SPF雏鸡血液和局部淋巴组织CD4+/CD8+细胞及相关细胞因子表达的影响

旨在探讨禽网状内皮组织增生病病毒（reticuloendotheliosis virus,REV）对SPF雏鸡血液和局部淋巴组织中T淋巴细胞数量以及相关细胞因子表达的影响。将96只1日龄SPF雏鸡随机分为REV感染组和对照组,应用流式细胞术、酸性α-醋酸萘酯酶染色（acid α-naphthyl acetate esterase,ANAE）、荧光定量PCR等方法对上述指标进行检测。试验数据表明,与对照组相比,感染组雏鸡血液CD4⁺T淋巴细胞数量在第7~35天显著降低、CD8⁺T淋巴细胞数量在第7~28天显著升高,CD4⁺/CD8⁺比值在第7~28天显著降低（均P<0.05或P<0.01）;局部淋巴组织哈德尔腺（Hader’s gland,HG）、派伊尔结（Peyer’s patch,PP）和盲肠扁桃体（caecal tonsil,CT）中ANAE⁺T淋巴细胞数量均显著降低（均P<0.05或P<0.01）;GH、PP和CT中细胞因子IL-2、IFN-γ和TNF-α 转录量都有不同程度升高。本研究表明,REV感染引起雏鸡血液中CD4⁺ T淋巴细胞数量降低、CD4⁺/CD8⁺T淋巴细胞比例失衡、局部淋巴组织中T淋巴细胞数量相对减少及细胞因子TNF-α转录持续升高与REV造成感染雏鸡细胞免疫功能显著降低密切相关。     

Lymphocyte subsets in peripheral blood of dogs--a flow cytometric study

Slight differences in the results of papers describing lymphocyte subsets distribution in the peripheral blood of healthy dogs may be explained by differences in monoclonal antibody clones and sources, breed and age of animals examined, methods of sample treatment, or methods of result analysis. In this paper, we described the effect of sample processing and of sample storage as well as the effect of age, breed, and gender of dogs on lymphocyte subset distribution. No significant differences were found between samples processed following a whole-blood lysis method and samples processed after density gradient separation. Furthermore, no significant differences were found between samples processed within 2h after collection and those stored at 4 degrees C for 12-16 h before processing. Age-related changes were evident in lymphocyte subset distribution in the peripheral blood of 38 Beagles divided according to their age into the six groups: (1) 5-6 days; (2) 2 months; (3) 6 months; (4) 1-2 years; (5) 3-5 years; and (6) >5 years. The percentage of B-lymphocytes (CD21-like positive cells) in the peripheral blood of newborn pups was 39.5+/-5.7 and decreased with advancing age. The percentage of CD8+ lymphocytes was 7.7+/-3.4 after birth and increased with advancing age. No age-related changes were observed in the percentages of CD4+ lymphocytes. The CD4+:CD8+ ratio decreased with advancing age. No significant age-related change was observed for lymphocytes bearing the gammadelta-TCR. Some breed differences were evident. Adult (1-5-year-old) Beagles, German Shepherds, Dalmatians, and Dachshunds were examined. The percentages of lymphocytes were higher in Beagles and Dachshunds than in Dalmatians and German Shepherds. The highest and the lowest absolute lymphocyte counts were found in Beagles and German Shepherds, respectively. As a consequence, German Shepherds showed the lowest absolute counts of the individual lymphocyte subpopulations and the widest neutrophil:lymphocyte ratio. Dalmatians showed the lowest percentage of CD3+ cells, the highest percentage of CD21+ cells, and the lowest CD4+:CD8+ ratio. German Shepherds showed the lowest percentage of CD21+ cells and the highest CD4+:CD8+ ratio. Females in Beagles and Dachshuns had nonsignificantly higher percentages of total lymphocytes, CD3+, CD4+, and nonsignificantly lower percentages of CD21+ lymphocytes. We concluded that there are age-, breed-, and perhaps also gender-related differences in lymphocyte subset distribution in the peripheral blood of dogs. Therefore, there is need to use appropriate control group in the experimental protocols. Among-breed differences could explain, at least partly, breed predisposition for some diseases.     

Differences in the kinetics of T cell accumulations in C3H/HeN (Bcg-resistant) and C57BL/6 (Bcg-susceptible) mice infected with Mycobacterium paratuberculosis

The accumulation of various T cell subsets in Bcg-susceptible (C57BL/6) and -resistant (C3H/HeN) strains of mice were compared following an intraperitoneal infection with Mycobacterium paratuberculosis. Groups of mice from both strains were killed at 3, 5, 10, 15, 30, and 150 days after infection and lymphocytes were harvested from the peritoneal exudate cells (PEC), spleen, intestinal epithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches, and mesenteric lymph node (MLN) and labelled with monoclonal antibodies to CD3, CD4, CD8, γδ TCR, CD25, and CD44 for flow cytometric analysis. Uninfected C3H/HeN mice had higher proportions of CD4+ cells in the spleen, MLN, LPL, IEL and Peyer's patches, while uninfected C57BL/6 mice had higher proportions of CD8+ and/or γδ T cells. Significant increases in accumulation of CD8+ and γδ T cells were detected in the peritoneum and other tissues in both strains of mice after infection. Higher CD4/CD8 ratios were observed in most lymphoid tissues of C3H/HeN mice, while increased proportions of CD8+ and/or γδ T cells were present in C57BL/6 mice. These results indicate that significant differences in T cell profiles exist between these two strains of mice, both inherently and in response to infection with M. paratuberculosis. Innately lower levels of CD4+ cells and/or higher percentages of CD8+ and γδ T cells may play a role in the increased susceptibility of C57BL/6 mice to infection with M. paratuberculosis.     

Changes in Distribution and Numbers of CD4+ and CD8+ T‐lymphocytes in Lymphoid Tissues and Intestinal Mucosa in the Early Phase of Experimentally Induced Early Onset Mucosal Disease in Cattle

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune‐mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post‐inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post‐inoculation was paralleled by a decrease of B‐lymphocytes and an increase of CD4+ T‐lymphocytes. An increased number of CD8+ T‐lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T‐lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T‐lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T‐lymphocytes in sites with lesions. This might indicate a cell‐mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T‐lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.     

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a 'memory' phenotype, being CD62L(-)/CD45RA(-) and expressing high levels of CD2 and CD11a; efferent cells are largely 'na?ve', being CD62L(+)/CD45RA(+) with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNgamma, TGFbeta and TNFalpha) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments.     

Changes in lymphocyte subsets in the bronchus-associated lymphoid tissue of goats naturally infected with different Mycoplasma species

The distribution of cells containing lysozyme, S-100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus-associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3-5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1-6), M. mycoides ssp. capri (goats no. 7-12) and M. capricolum ssp. capricolum (goats no. 13-18). Microscopically, infected animals showed a moderate broncho-interstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3+ T lymphocytes, and the ratio of CD4+:CD8+ cells was > 2. The BALT showed large germinal centres mainly composed of IgG+ B lymphocytes, with numerous S-100+ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG+ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.