Experimental haemonchosis in goats: effects of single and multiple infections in the host response

Histopathological changes and the distribution of T lymphocytes (CD3), B cells (CD79alpha) and IgG secreting plasma cells were recorded in the abomasum and abomasal lymph nodes of goats during early and late post-infection stages with one to four doses of Haemonchus contortus L3. The infiltration of eosinophils, mast cells, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells in the abomasal mucosa increased dramatically from 10dpi onwards, whereas globule leukocytes were observed only during chronic infection. In late post-infection stages abomasal infiltration of globule leukocytes, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was significantly higher (P<0.05) in reinfected (groups 6-8) than in primarily infected goats (group 5). In the abomasal lymph nodes, marked hyperplasia of lymphoid follicles and medullary cords, with increase of CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was recorded from 10dpi (group 3) onwards. Worm burdens and the severe abomasal response during the late post-infection stages suggests that a rapid expulsion of nematodes did not occur. The prolonged time required for generating globule leukocytes suggested that immune mechanisms dependent of this cell type are of crucial importance in the protective immunity against H. contortus in goats.     

Bovine dendritic cells generated from monocytes and bone marrow progenitors regulate immunoglobulin production in peripheral blood B cells

We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4(+) T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4(+) T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.     

Humoral and cellular immune responses in dogs with inapparent natural Leishmania infantum infection

Molecular analysis, serology and immunophenotyping for T lymphocytes and their subsets, B lymphocytes and monocytes were performed on dogs naturally infected with Leishmania infantum. Animals were categorised as asymptomatic dogs I (AD-I), with negative serology and positive molecular results, and asymptomatic dogs II (AD-II), with positive serology and positive molecular results, and these were compared to symptomatic dogs (SD) and control dogs (CD). AD-I exhibited immunophenotypic features similar to those of CD, including isotype profiles and concentrations of monocytes. Similar biomarkers were found in AD-II and SD, such as, higher levels of immunoglobulins IgG, IgG2, IgM and IgA and higher concentrations of eosinophils. High frequencies of T lymphocytes and CD4(+) T cells were observed in both AD-I and AD-II compared to SD, whereas CD8(+) T cells were higher only in AD-II compared with SD. Analysis of B lymphocytes revealed an increased frequency of this cell type only in AD-II animals compared with SD. Asymptomatic dogs appear to have a dichotomous infection spectrum that can influence the humoral and cellular immunological status during canine visceral leishmaniasis.     

Numbers and distribution of immune cells in the tunica mucosa of the small and large intestine of full-thickness biopsies from healthy pet cats

In this study, CD3(+) T lymphocytes and IgA(+) , IgG(+) and IgM(+) plasma cells were quantified in the tunica mucosa of the intestinal tract of 12 pet cats without gastrointestinal diseases. The study included full-thickness biopsies of the duodenum, jejunum, ileum and colon. The distribution and quantification of CD3(+) T cells, IgA(+) , IgG(+) and IgM(+) plasma cells within the intestinal tunica mucosa was performed by using immunohistochemical methods and computer-aided morphometry. CD3(+) T cells were significantly prominent in the villi and their numbers increased from duodenum to ileum but decreased towards the colon. The predominant type of plasma cells was IgA(+) cells, followed by IgM(+) cells. The number of IgG(+) cells was generally low compared to the other plasma cell types investigated. The results of the vertical distribution showed that IgA(+) and IgM(+) plasma cells were most numerous in the lower crypt areas, whilst IgG(+) plasma cells accumulated in the upper crypt region with a decline towards the villi and the lower crypt areas of control cats. All types of plasma cells showed a general decline from the duodenum towards the caudal sections of the intestinal tract regarding the horizontal distribution of plasma cells. This study provides a comprehensive overview on the vertical and horizontal distribution and the number of CD3(+) T cells and IgA(+) , IgG(+) and IgM(+) plasma cells in the intestinal tunica mucosa of pet cats.     

Changes in peripheral lymphocyte subsets in the type 1 diabetic dogs treated with insulin injections

Plasma metabolites and peripheral lymphocyte subsets were measured in ten diabetic and ten control dogs to investigate their significances as indicators to evaluate immune states in the diabetic dogs. Diabetic dogs were treated with insulin injections, however their plasma glucose and fructosamine concentrations were significantly higher than those of the controls. There were no significant differences in counts of total white blood cells (WBC) and lymphocyte CD8(+) cells (cytotoxic T cells) between the control and the diabetic dogs. In the diabetic dogs, the counts of CD3(+) (T cells), CD4(+) (Helper T cells) and CD21(+) (B cells) cells and the peripheral lymphocytes CD4/CD8 ratio were significantly lower than those in the control dogs. We confirmed abnormality of lymphocyte subsets in insulin treated diabetic dogs and it may relate to depression of immunocompetence and high susceptibility to common infectious diseases.     

Experimental Ehrlichia canis infection in the dog does not cause immunosuppression

A carrier state develops in some Ehrlichia canis-infected dogs due to ineffective host defenses. The subsequent development of immune-mediated diseases or opportunistic infections in chronic ehrlichiosis suggests dysregulation of immunity; however, the immunobiology of this infection has not been well characterized. In this study, eight dogs were infected with E. canis, and changes in seroreactivity, serum immunoglobulin (Ig) concentrations, peripheral blood T cell subsets, lymphocyte blastogenesis (LBT), and lymphokine-activated killer (LAK) activity were evaluated over 4 months. Infection, which was documented by seroconversion, polymerase chain reaction, and blood culture, caused self-limiting fever and thrombocytopenia. Infected dogs developed an anti-E. canis antibody response but were not immune to re-infection. Serum IgM, IgG, and IgA concentrations were unaffected by E. canis. The percentage of circulating CD4(+) T cells was similar in uninfected and infected dogs at all points. Infected dogs developed a CD8(+) lymphocytosis 6 weeks after inoculation that subsequently subsided, despite organism persistence. Functional defects of cell-mediated immunity, measured as suppression of LAK activity or mitogen-driven LBT, were not observed. These results suggest that immune responses are not grossly impaired in young dogs during the first several months following experimental E. canis infection.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.     

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.     

Histological studies on the ontogeny of bovine palatine and pharyngeal tonsil: germinal center formation, IgG, and IgA mRNA expression

The development and distribution of lymphocyte subsets in calf palatine and pharyngeal tonsil were examined. During prenatal development, B cells were distributed in the subepithelial area, and T cells and MHC class II⁺ cells were found in the deep layer of B-cell area, respectively, in both tonsils. At neonatal stage, lymphoid follicle containing a few CD4⁺ cells have been formed in both tonsils. IgG⁺ and IgA⁺ cells were found in the parafollicular and epithelial area. At 3 months old, many germinal centers were recognized in both tonsils. CD4⁺ cells and IgG mRNA expression were detected in light zone of germinal centers. Many IgG, and IgA mRNA expressions also could be detected in the parafollicular and subepithelial area of both tonsils. The data suggest that both tonsils have an important role of local immune defense against invading antigen after birth. The comparison of the histological characteristics of tonsil and Peyer's patch during ontogeny is also discussed.     

Expression characteristics of immune factors in the yak (Bos grunniens) testis

The goal of this study was to characterize and evaluate the main markers of macrophages, T lymphocytes, B lymphocytes and plasmocytes in the testis of juvenile and adult yaks by quantitative real-time polymerase chain reaction and immunohistochemistry. Within the same age group, the mRNA expression of CD68 was always highest, followed by that of CD3ε, CD79α, IgG and IgA. Moreover, CD68, CD3, CD79α, IgA and IgG positive cells were all located in the testicular interstitial tissues of juvenile and adult yaks. In the same age group, the frequency of CD68 positive macrophages was higher than that of CD3 positive T lymphocytes, which was followed by that of CD79α positive B lymphocytes and IgA and IgG positive plasmocytes. No significant difference was observed between the B lymphocyte and plasmocyte frequencies in yak testes. Furthermore, CD68, CD3ε, CD79α, IgA and IgG mRNA expression levels and the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells increased from juveniles to adults. Similarly, the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells also increased with age. These results suggest that in the yak testis, the immune defence system against pathogens might primarily comprise macrophages and T lymphocytes in the testicular interstitial tissue. Moreover, the testicular immune environment may mature and expand to a fully functional state in adult yaks. The low frequencies of B lymphocyte and plasmocyte in yaks, differing from those in rodents and humans, might be related to the fact that yaks live in low-oxygen plateaus.     

Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.     

Lymphocyte subsets in the mammary gland of sows

The presence and localisation of lymphocyte subsets together with class II bearing cells in the mammary gland of sows, were studied at different periods of the reproductive cycle by immunohistochemistry and compared with blood. All cell types involved in the immune response were present in the mammary gland at the different stages of gestation and lactation and nearer the alveolar epithelium as gestation proceeded: T lymphocytes, including CD4+ and CD8+, B lymphocytes and class II bearing cells (epithelial cells and macrophages). The results indicated an early accumulation of T lymphocytes, specifically T helper cells, during pregnancy; the specific increase of IgA lymphocytes occurring after this phase could suggest a role for these T cells in the induction of IgA response. The local accumulation of immune cells sustains the view that the mammary gland is able to mount a true local immune response and the increase in CD8+ cells near the epithelium suggests a role in local immune defence.     

Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens

A novel Salmonella Enteritidis (SE) vaccine candidate strain, JOL919 was constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. The study was carried out to evaluate the strain as a vaccine candidate against salmonellosis. The strain showed the enhanced macrophage invasion, early bacterial clearance and higher immune responses as compared to the other mutants, JOL917 (Δlon) and JOL918 (ΔcpxR), and the wild type. In further analysis, the chickens immunized with JOL919 showed a significant increase in plasma IgG and intestinal secretory IgA levels, which was an indication of robust humoral and mucosal immune responses induced by the candidate. The lymphocyte proliferation response and CD45(+)CD3(+) T cells, associated with an activation of T helper and cytotoxic cells, were also significantly increased in the immunized group, which indicated that the candidate also induced cellular immune responses. The immune cell influx into caecal tissues analyzed by immunohistochemistry showed that CD8(+) T cells were predominated in the immunized group, suggesting that the candidate can clear the invaded pathogen in the intestines by a more direct way involving cytotoxic activity. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.     

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.     

Characterization of immune cell infiltration into canine intracranial meningiomas

Meningiomas are the most common intracranial tumors in dogs. A variety of inflammatory cells have been shown to invade these tumors in people, but little is known about interactions between the immune system and naturally occurring brain tumors in dogs. The purpose of this study was to investigate the presence of a variety of immune cell subsets within canine intracranial meningiomas. Twenty-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry with antibodies specific for CD3, CD79a, CD18, CD11d (αD), CD45RA, forkhead box P3, and Toll-like receptors 4 and 9. Immune cell infiltration was evident in all samples, with a predominance of CD3(+) T cells. Large numbers of CD18(+) microglia and macrophages were noted surrounding and infiltrating the tumors, and a subset of these cells within the tumor appeared to be CD11d(+). Scattered macrophages at the tumor-brain interface were TLR4(+) and TLR9(+). Rare CD79a(+) B cells were noted in only a small subset of tumors. Lesser numbers of lymphocytes that were CD11d(+), CD45RA(+), or FoxP3(+) were noted in a number of the meningiomas. Although the function of these cells is not yet clear, work in other species suggests that evaluation of this immune cell infiltrate may provide important prognostic information and may be useful in the design of novel therapies.     

Postnatal development of leukocyte subset composition and activity in dogs

The aim of the presentation is to summarise our data on the counts and activity of circulating canine leukocytes at birth and on their changes in the first 3 months of life. On day 1, neutrophil counts were almost three times higher than lymphocyte counts. During the first week of life, a decrease of neutrophil and an increase of lymphocyte counts, resulting in a predominance of lymphocytes, were observed. Neutrophil counts reached values comparable with those in adults in 1 month. Lymphocyte counts were higher than those in adults during the first 3 months. From birth to the age of 3 months, the phagocytic activity of neutrophils was nonsignificantly higher than in young adults. When compared with adults, the peripheral blood of new-born pups contained a lower proportion of T lymphocytes (detected by CD3 and CD5 markers), with a very low percentage of CD8(+) cells and a higher proportion of CD21(+) B lymphocytes. The counts of individual subsets levelled out during the first 3 months of life, although the proportion of CD21(+) B cells remained higher all the time. Lymphocytes of new-born pups were able to respond to nonspecific mitogen stimulation. Spontaneous proliferation in vitro was higher during the first week of life. Although in vitro stimulation of lymphocytes with Concanavalin A in some pups was comparable with that of adult dogs, mean activity was weaker. Pups with zero or very low levels of maternal antibodies were able to develop specific immune responses to a parvovirus antigen as early as at 2 weeks of age. On the basis of these data, we assume that pups are born with an immune system that can respond to external stimuli. Nevertheless its development continues in the postnatal period and some parameters differ from adult values for at least 3 months after birth.     

In situ characterization of the granulomatous immune response with time in nonhealing lesional skin of Leishmania braziliensis-infected rhesus macaques (Macaca mulatta)

We have recently introduced a macaque (Macaca mulatta) model of Leishmania braziliensis-induced self-healing cutaneous leishmaniasis in which the T cell-mediated inflammatory response effectively promotes parasite clearance and granuloma resolution. Here we show that macaques infected with a highly pathogenic L. braziliensis strain displayed longstanding granulomatous lesions which lasted until the end of the observation period (52 weeks). Immunoperoxidase staining of representative tissue sections indicated that distinct cell populations (CD3, CD4, CD8, CD20, Foxp3, CD20, CD68, HLA-DR, CCL2, and CXCL-10) change uniformly during infection, suggesting that the same components of the local immune response are working in unison. This model also confirmed that granuloma formation is orchestrated by diverse inflammatory mediators that are important for T helper type 1 (Th1) cell development and macrophage effector functions. Cytometry analysis of ex vivo granuloma-derived leukocytes revealed accumulation of distinct functional subsets of effector and regulatory T cells into the inflamed skin. We provide evidence that local interleukin (IL)-10 production by both Foxp3(+) and Foxp3(-) CD4(+) T subsets is likely important in promoting lesional granuloma maintenance. Further studying the immune suppression mechanisms that induces granulomas in L. braziliensis-infected macaques may reveal new opportunities for therapeutic control of this important human disease.     

免疫细胞在牦牛附睾和输精管的分布规律

旨在研究免疫细胞在健康雄性牦牛附睾和输精管的分布。采用免疫组织化学和实时荧光定量(qRT-PCR)方法对幼龄(5~6月龄)及成年(3~4岁)牦牛附睾(头、体、尾)和输精管中CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞的分布特征及其表面标志分子的表达水平进行研究。结果显示：CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞主要分布在附睾管和输精管的上皮和间质;另外,CD68和CD3 mRNA和蛋白水平在各年龄组牦牛附睾头和附睾体显著高于附睾尾和输精管(P < 0.05),而CD79α、IgA和IgG mRNA和蛋白水平在附睾尾和输精管显著高于附睾头和附睾体(P < 0.05);此外,在成年牦牛附睾和输精管CD3、CD79α、IgA、IgG、CD68 mRNA和蛋白水平均显著高于幼龄牦牛(P < 0.05)。综上提示,牦牛附睾头可能主要是细胞免疫发生的位点,而附睾尾和输精管则主要进行体液免疫应答,此外,成年牦牛附睾和输精管的局部免疫可能更完善,以上数据为进一步研究高原牦牛局部生殖免疫和病理提供了形态学资料。     

Phenotypic comparison of ileal intraepithelial lymphocyte populations of suckling and weaned calves

Ileal intraepithelial lymphocyte (IEL) suspensions from suckling calves (1-3 weeks old) and weaned calves (3-6 months old) were phenotyped to determine whether there were differences in the lymphocyte populations consistent with postnatal maturation of the mucosal immune system. Flow cytometric comparisons of IEL from the two age groups revealed the presence of significantly larger proportions of CD4+ T lymphocytes and CD8+ T cells in the weaned animals. In contrast, there was a significantly larger proportion of B-B2+ IEL in the suckling calves. Freshly isolated IEL from both groups of calves expressed mRNA for TNF-alpha and IFN-gamma, but not IL-4 or IL-10. The B-B2+ IEL population was more closely examined by flow cytometry. These cells co-expressed IgM and CD21. However, they did not express IgA, IgG1, nor any of several additional leukocyte differentiation molecules. Immunohistochemical data confirmed the presence of IgM+ lymphocytes, and the paucity of IgA+ and IgG1+ lymphocytes in suckling calf ileum. However, substantial numbers of IgA+ and IgG1+ cells were observed in weaned calf ileum. Together, the data are consistent with ongoing postnatal maturation of the gut mucosal immune system.