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.     

Influence of bestatin, an inhibitor of aminopeptidases, on T and B lymphocyte subsets in mice

Bestatin, a low-molecular weight dipeptide, is a potent inhibitor of aminopeptidase N which has been demonstrated to have antitumor and immunomodulatory effects. The effects of bestatin (10, 1 and 0.1 mg/kg) administered intraperitoneally once, five or ten times to mice on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes were studied. It has been found that bestatin administered ten times at doses of 10, 1 and 0.1 mg/kg increased the total number of thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes. Bestatin also changed the percentage and the absolute number of T cell subsets in the thymus and T and B lymphocytes in the peripheral lymphatic organs. Five and ten exposures to bestatin (10, 1 and 0.1 mg/kg) increased the absolute count of both immature CD4+CD8+ and CD4-CD8- thymic cells. Moreover, both a single and multiple administration of bestatin (1 and 0.1 mg/kg) decreased the percentage and absolute count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Both a single and multiple administration of bestatin at all the doses under investigation augmented the percentage and the absolute count of CD19+ (B lymphocytes) in the peripheral lymphatic organs. The results of the study show that there is a relationship between the effect induced by bestatin and the dose of the drug as well as the number of doses applied. The strongest effect on the T and B lymphocyte subsets was noted after five injections of bestatin at doses of 1 and 0.1 mg/kg.     

Lymphatic organ development in dogs: major lymphocyte subsets and activity

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

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

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

Changes in mouse circulating leukocyte numbers in C57BL/6 mice immunosuppressed with dexamethasone for Cryptosporidium parvum oocyst production

The Iowa strain of Cryptosporidium parvum will not propagate in immunocompetent mice, but will successfully infect genetically immunocompromised nude or SCID mice as well as immunocompetent mice which have been immunosuppressed with glucocorticoids. Using dexamethasone-tetracycline is one published method for immunosuppressing mice for the production of C. parvum oocysts. However, dexamethasone-induced immunosuppression is variable, because it is dependent on the total daily water consumption of each individual mouse. The changes in circulating leukocytes and other immune system associated organs before, during and after dexamethasone suppression were analyzed for comparison with a new single injection methylprednisolone acetate (MPA) suppression model. The dexamethasone-induced immunocompromised state was associated with a greater than 90% sustained drop in circulating T-lymphocytes, a greater than 700% increase in circulating mature segmented neutrophils and a severe depletion of circulating monocytes. The thymus and spleen decreased in size by over 80%. Oocyst shedding in suppressed mice started within 4 days of oocyst inoculation and persisted for 6 days post-dexamethasone treatment. Seven days after dexamethasone withdrawal, circulating neutrophils still were 549% higher than controls. Circulating CD3 and CD4 lymphocytes remained depressed by 85-90% while on dexamethasone and for 7 days after discontinuing dexamethasone. CD8 lymphocyte numbers initially decreased by 90%, but rose even while on dexamethasone and even with severe thymic involution. At day 7 post-dexamethasone treatment, the spleen was 119 mm(3), approximating the same size as controls. Fourteen days post-dexamethasone treatment, which was 8 days after oocyst shedding had ceased, the CD8 counts per 5000 events were only 1.6% below controls, while the CD3 and CD4 counts were still depressed by 66%. The thymus now was about one quarter smaller than the controls. The rise in circulating CD8 lymphocytes, when oocyst production stopped, suggests that CD8 positive lymphocytes may play a significant role in vivo in clearing the parasite. The overall pattern of immunosuppression was nearly identical to that observed with the methylprednisolone acetate immunosuppression model.     

Alterations of lymphocyte subpopulations in healthy dogs with aging and in dogs with cancer

Changes in an individual's immune status are considered major contributing factors towards the morbidity of cancer and mortality of aging. To evaluate age-related changes in the immune status of dogs, the immunophenotypes (CD3, CD4, CD8 and CD21) of peripheral blood lymphocytes were measured in 160 healthy dogs aged from 1 to 17 years, and in 365 dogs with various tumors and at various stages. In healthy dogs, the absolute numbers of white blood cells, lymphocytes, and CD3(+), CD4(+) and CD21(+) lymphocytes decreased significantly with age. The relative percentages of lymphocytes and CD4(+) cells decreased significantly, while CD8(+) cells increased significantly with age. The CD4:CD8 ratio showed a significant age-related decrease. In contrast, dogs with tumors possessed significantly lower absolute numbers and relative percentages of all lymphocyte phenotypes, while the CD4:CD8 ratio was significantly higher than in age-matched controls. The relative percentages of CD3(+) and CD8(+) lymphocytes were significantly lower in dogs with distant metastases compared with dogs without metastases, and the CD4:CD8 ratio increased with advanced stage. These observations illustrate the significant changes in immune status with age and the presence of marked immunological defects in a large-scale study of dogs with advanced tumors.     

Primary and anamnestic responses of bovine bronchoalveolar and peripheral blood lymphocyte subsets to aerosolized Pasteurella haemolytica A1

Site-specific responses of bronchoalveolar and peripheral blood lymphocyte subsets were compared during primary and anamnestic immune responses against live Pasteurella haemolytica A1 (Ph1). Eight 1-year old calves were sequentially exposed intrabronchially with aerosolized Ph1 on days 0, 14, and 21, and two calves were sham exposed. Bronchoalveolar and peripheral blood lymphocytes were analyzed before each Ph1 exposure, and on days 3 and 7 post exposure using single and two-color flow cytometry to identify CD2+, CD4+, CD8+, CD21+, CD45R+, CD25+ and gammadelta lymphocyte subsets. Significant differences (p < 0.05) in bronchoalveolar and peripheral blood lymphocyte subsets were observed before Ph1 exposure. Subsequent aerosol exposures, resulted in significant (p < 0.05) changes in bronchoalveolar lymphocyte subsets and the CD4:CD8 bronchoalveolar lymphocyte ratio, but concomitant changes were not observed in peripheral blood lymphocytes. Expression of CD2, CD4 and CD8 lymphocyte differentiation antigens was consistently lower and more heterogeneous on bronchoalveolar lymphocytes. Differential analysis of bronchoalveolar leukocytes revealed a significant increase in bronchoalveolar lymphocytes and neutrophils during anamnestic responses.     

Effects of road transportation on lymphocyte subsets in calves

The effect of transportation on peripheral blood lymphocyte subsets in 24 calves was investigated by flow cytometry. Blood was collected before departure, on arrival, at 24h and 1 week after arrival. Highest leucocyte and neutrophil counts, associated with increased concentrations of cortisol and catecholamines, indicated that stress was maximal upon arrival. At this time, a decrease in the percentages of all T lymphocyte subsets was evident, while they did not decrease as absolute counts. The proportion of CD21(+) cells did not change, indicating that the relative reduction of T lymphocyte subsets was not related to an increase in B lymphocytes. These variations may be due to the increase of a natural killer (NK) cell subset. NK cell expansion, together with increasing lymphocyte count and increasing major histocompatibility complex class II expression, may indicate stress-induced stimulation of the immune system.     

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.     

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.     

AA肉鸡免疫禽流感油乳剂灭活疫苗细胞免疫应答的变化

为了解肉鸡生长期内免疫前后的细胞免疫反应,本实验采用CD3/CD4/CD8三色流式细胞术、MTT法和Griess法依次检测了AA肉鸡免疫禽流感灭活疫苗和非免疫状态下外周血、胸腺和脾脏中T淋巴细胞及其亚群的相对含量、外周血中T淋巴细胞对PHA-P的增殖反应能力和诱导巨噬细胞NO分泌量的变化。结果表明,非免疫状态下,AA肉鸡外周血CD4+、CD8+、CD3+T淋巴细胞相对百分含量在7日龄时均为最低,脾脏中CD4+T淋巴细胞相对百分含量在整个试验期一直呈下降趋势,免疫后外周血、脾脏和胸腺中CD4+、CD8+、CD3+T淋巴细胞相对百分含量均明显高于非免疫组,且脾脏CD4+T淋巴细胞呈阶段性上升状态;各时期淋巴细胞刺激指数(SI)值和巨噬细胞NO分泌量检测结果表明,免疫组均高于同期对照组(p<0.05)。研究结果还表明:肉鸡在7日龄时细胞免疫水平最低,灭活疫苗可以激发肉鸡产生较强的细胞免疫应答。     

Evolution of the peripheral blood lymphocyte populations in multiparous rabbit does with two reproductive management rhythms

The emergence of epizootic rabbit enteropathy is leading to changes in weaning protocols in commercial rabbitries. Traditional weaning protocols are being replaced with late weaning, beyond 35 days postpartum (dpp). The main objectives of this study were to compare the peripheral blood lymphocyte populations of multiparous rabbit does under two reproductive rhythms (insemination at 11 dpp and weaning at 28 dpp, insemination at 25 dpp and weaning at 42 dpp), and to assess the influence on those of kits. Samples of peripheral blood were taken in 22 adult females and 44 of their kits at different critical times, and several lymphocytic populations were evaluated by flow cytometry. Additionally, the perirenal fat thickness of does was also measured at partum and weaning to observe if body condition correlates with lymphocyte populations. During whole lactation, counts of total, CD5(+), CD4(+) and CD8(+) lymphocytes of females were generally lower with weaning at 42 dpp compared to 28 dpp. Moreover, counts of total, B and CD5(+) lymphocytes in rabbit does weaned at 42 dpp correlated to their body condition (+0.60 to 0.82; P<0.05), contrary to that observed in rabbit does weaned at 28 dpp. Some correlations between lymphocyte counts in both groups of does and weaning rabbits were observed. At weaning, those young rabbits weaned at 42 dpp had a significantly lower number of CD4(+) lymphocytes than those weaned at 28 dpp (P<0.01). In conclusion, the 42 ddp rabbit does presented a lower number of total lymphocytes and lymphocytic subpopulations during lactation and at weaning, as well as lesser capacity of adjustment during the gestation-lactation cycle.     

Analysis of leucocytes and lymphocyte subsets in cats with naturally-occurring cryptococcosis but differing feline immunodeficiency virus status

SUMMARY: Although cryptococcosis is a well-characterised disease of cats, the factors predisposing individuals to infection are unknown. As an indication of the immune status of an individual, lymphocyte subsets can be analysed. Reference ranges for feline lymphocyte subsets (Pan T+, CD4+, CDS+ and B cells) were established using a rapid whole blood technique and flow cytometry. There were no effects of age or sex on lymphocyte subset values. The numbers of circulating leucocytes and lymphocyte subsets were determined in FIV-positive and FIV-negative cats with cryptococcosis and compared with a group of healthy control cats.
There were only minor differences in the numbers of lymphocyte subsets among the subgroups of cats examined in the study and the predisposition to cryptococcosis in cats could not be explained by deficiencies in lymphocyte subsets. There was a tendency for FIV-negative cats with cryptococcosis to have reduced numbers of circulating CD4+ cells and lower CD4:CD8 ratios compared with normal cats, although the interpretation of this finding was complicated by the wide reference range for normal cats. The extent to which this is the cause of the fungal infection was not determined.
The only difference in leucocyte or lymphocytes subset values between FIV-negative cats with cryptococcosis and FIV-positive cats with cryptococcosis was that the CD4+ percentage was lower in the FIV-positive cats. The absolute CD4+ count was similar however, in FIV-positive and FIV-negative cryptococcosis cases. On the basis of this and other available information, the categorisation of cryptococcosis as a disease defining the AIDS phase of FIV infection may be incorrect.     

Age-related changes in lymphocyte subsets of quarter horse foals

OBJECTIVE: To characterize changes in lymphocyte subsets over time in foals from birth to 18 weeks of age, accounting for differences among individuals, and to determine the effect of overnight storage of blood samples on foal lymphocyte subset concentrations. ANIMALS: 8 healthy Quarter Horse foals from birth to 18 weeks of age. PROCEDURE: Blood samples were collected longitudinally from birth to 18 weeks of age and a CBC performed on each sample. The samples were stained for lymphocyte markers, either immediately or after overnight storage and analyzed by flow cytometry. RESULTS: Total leukocytes, total lymphocytes, and the absolute concentrations of all lymphocyte subsets increased significantly with age. The proportions of B29A+, CD21+, and-equine major histocompatability complex class-II molecule+ lymphocytes increased significantly with age. The proportion of equine (Eq) CD5+, EqCD8+, and EqWC4+ lymphocytes decreased significantly with age. Significant differences among foals were found with respect to initial concentrations with respect to initial concentrations, but not with respect to the rate of increase of the various subsets tested. Significant differences were not found in subset values when comparing blood samples stained on the day of collection or after overnight storage at room temperature (approx 21 C) or under refrigeration. CONCLUSIONS AND CLINICAL RELEVANCE: These results are consistent with an increase in subset numbers and proportions over time, but with individual differences among foals. The observation of individual differences in subsets among foals suggests that there may be individual differences in susceptibility to infectious disease during the perinatal period. The absence of an effect of overnight storage makes field studies of lymphocyte subset concentrations more feasible.     

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.     

Early-life longitudinal survey of peripheral blood lymphocyte subsets in Beagle dogs

A longitudinal study of peripheral blood lymphocyte subsets from 7 to 15 months of age was performed in Beagle dogs employing a multiparametric flow cytometry. The data were compared with data obtained from adult Beagle dogs that were housed in the same animal facilities and that were subjected to the same controls during the 34 weeks of the study. Absolute counts of total lymphocytes and CD3+ T, CD3+CD4+ Th and CD21+ B lymphocytes decreased during the entire 34 weeks period of the study in the young dogs group. The same was observed with regard to the percentage of CD3+CD4+ Th lymphocytes and the CD4/CD8 ratio, while the percentage of CD3+CD8+ Tc lymphocytes increased from 7 to 15 months of age. These age-related changes found in lymphocyte subsets distribution of young dogs led to level the absolute and relative values of adult dog lymphocytes. The observations of this longitudinal study illustrate the changes related to maturation of lymphocyte subsets that occur during early life in Beagle dogs.     

Influence of betulinic acid on lymphocyte subsets and humoral immune response in mice

Betulinic acid is a pentacyclic triterpene found in many plant species, among others, in the bark of white birch Betula alba. Betulinic acid was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial, anticancer and anti-inflammatory activities. The effects of betulinic acid (50, 5, 0.5 mg/kg) administered orally five times at 24 hours intervals to non-immunized and red blood cells (SRBC)-immunized mice were determined. The present study examined the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, and the percentage of subsets of T cells (CD4+CD8+, CD4CD8, CD4+, CD8+) in thymus,T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes, as well as white blood cell (WBC) and differential leukocyte counts in non-immunized mice, and humoral immune response in SRBC-immunized mice. SRBC was injected 24 hours after administration of the last dose of betulinic acid. It was found that betulinic acid administered orally five times at the dose of 0.5 mg/kg increased the total number of thymocytes, splenocytes, lymphocytes of mesenteric lymph node cells, and the weight ratio of the spleen and mesenteric lymph nodes in non-immunized mice. Betulinic acid also changed the percentage of T cell subsets in the thymus and T and B lymphocytes in peripheral lymphatic organs. The effects of betulinic acid on T and B cell subpopulations depended on the dose applied. The strongest stimulating effect of betulinic acid was observed when the drug was administered at the dose of 0.5 mg/kg. Five exposures to betulinic acid (0.5 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells with corresponding increases in the percentage and absolute count of mature, single-positive CD4+ thymocytes and decreased the percentage and total count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Multiple administration of betulinic acid at the investigated doses augmented the percentage and absolute count of CD19+ cells in the peripheral lymphatic organs. Moreover, betulinic acid at the dose of 5 mg/kg administered prior to SRBC immunization increased the number of plaque forming cells (PFC) but decreased the production of anti-SRBC antibodies on day 4 after priming. Thus, betulinic acid is a potential biological response modifier and may strengthen the immune response of its host.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Characterization of a Cryptosporidium muris infection and reinfection in CF-1 mice

To establish control values for circulating cells and immune associated organs over the course of a self-limiting Cryptosporidium muris infection and rechallenge infection, mice were sacrificed at intervals starting before oral inoculation and ending after oocyst shedding had ceased. These values were used in other experiments to evaluate changes in these parameters induced by a single dose glucocorticoid immunosuppression model and in other immunosuppression studies. Flow cytometry counts of circulating T-lymphocytes and neutrophils, differential leukocyte counts, leukocyte morphology, spleen and thymus changes, and oocyst shedding were evaluated. Immediately after C. muris oocyst inoculation and up to the start of oocyst production (day 0 to day 7), the circulating blood profile showed a 50% drop in all leukocytes, including both large and small lymphocytes and CD3, CD4 and CD8 T-lymphocytes. There was an initial slight rise in circulating mature neutrophils after oocyst inoculation but numbers promptly dropped below normal and remaineded below normal. In the differential cell counts, monocytes with a fat, oval morphology increased by 60% at 24 h and remained high through oocyst shedding and beyond (day 8 through day 36). During oocyst shedding and continuing past the end of shedding, T-lymphocytes increased 100%. Monocytes with a flat, angular morphology increased in a similar manner. Immediately after oocyst inoculation the spleen contracted by 29%, but became 92% larger than its pre-inoculation size by day 14 when heavy oocyst shedding began. It remained enlarged through the end of oocyst shedding (day 29) and beyond (day 36). Spleen volume decreased and increased similar to changes in T-cell numbers. Throughout the C. muris infection the thymus remained largely unchanged. The transit of an oocyst bolus was followed from the stomach through the gut to the colon. No oocysts could be found in the stomach, caecum or feces of mice one half hour after oocyst inoculation. Likewise, an oral bolus of India ink passed from the stomach entirely into the colon after 3 h; therefore, no oocysts from the inoculum passed completely through the intestine and out into the feces. Recovered mice rechallenged with C. muris showed increased B-lymphocyte numbers; however, T-lymphocyte numbers remained level. The large lymphocytes increased after rechallenge, peaking on day 3, then decreased through day 10. B-cell numbers followed a pattern similar to the large lymphocytes. On day 10 of infection monocytes with a fat oval morphology rose sharply while B-cells fell in number. In both the initial infection and the rechallenge there was no unique blood profile which could definitely indicate a protozoal disease or identify a specific point during the course of the disease. There was no increase in the number of either small or large lymphocytes prior to increases in fat or flat monocytes.     

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.