Effect of intramammary infusion of beta-1,3-glucan or interleukin-2 on leukocyte subpopulations in mammary glands of sheep.

OBJECTIVE: To investigate effects of intramammary infusion of beta-1,3-glucan or recombinant ovine interleukin-2 (rOvIL-2) on blood and mammary leukocyte subpopulations and their expression of various surface antigens in sheep. ANIMALS: 12 healthy multiparous, nonpregnant, nonlactating fine-wool Merino ewes. PROCEDURE: Beta-1,3-glucan in pyrogen-free saline solution (PFSS; n = 6), rOvIL-2 in PFSS (3), or PFSS (3) was infused on days 0 and 7 in 1 udder half of each ewe. Jugular vein blood and mammary secretion samples were taken before infusion and on days 2, 7, and 21 after infusion. Total and differential leukocyte counts were obtained, and blood and mammary cells were labeled for flow cytometry. RESULTS: Slight swelling of the mammary glands was observed on day 2 after rOvIL-2 but not after beta-1,3-glucan infusion. Both substances induced significant increase in mammary secretion leukocyte numbers, compared with controls. Beta-1,3-Glucan induced an influx of monocyte/macrophages, whereas neutrophils were the predominating cell population after rOvIL-2 infusion. Beta-1,3-glucan induced selection for CD4+, B-cell+, WC1+, and L-selectin+ lymphocytes on day 14 after infusion. By comparison, rOvIL-2 induced selection for B-cell+ and L-selectin+ lymphocytes on days 14 and 21 and depletion of CD8 and, to some degree, of IL-2R+ lymphocytes. Beta-1,3-Glucan induced an increase in the proportion of CD14+ leukocytes, indicating selective migration of monocyte/ macrophages to the nonlactating udder. CONCLUSION: Beta-1,3-Glucan and rOvIL-2 can modulate nonspecific immunity in the udder of sheep but may exert their effects by differing mechanisms. Clinical Relevance-Stimulation of the nonspecific defense against udder infections may improve control of mastitis.     

Expression of CD3 and CD11b antigens on blood and mammary gland leukocytes and bacterial survival in milk of cows with experimentally induced Staphylococcus aureus mastitis.

OBJECTIVES: To differentiate early (1 to 8 days) from late (9 to 14 days) inflammatory phases and assess relationships between leukocyte phenotype and bacterial recovery in cows with Staphylococcus aureus-induced mastitis. ANIMALS: 10 first-lactation Holstein cows. PROCEDURE: Blood and milk samples were collected from 4 or 6 cows before and after intramammary infusion of sterile broth or S. aureus, respectively. Flow cytometric expression of CD3 and CD11b antigens on blood and milk leukocytes, leukocyte differential counts, bacterial counts in milk, and somatic cell counts were determined longitudinally. RESULTS: Density of CD3 molecules decreased on blood lymphocytes and increased on milk lymphocytes after infusion of bacteria. Density of CD11b molecules on lymphocytes and phagocytes and percentage of CD11b+ lymphocytes in milk increased significantly after infusion; maximum values were achieved during the early inflammatory phase. Density of CD3 and CD11b molecules on milk lymphocytes and macrophages, respectively, 1 day after inoculation were negatively correlated with bacterial recovery on day 1 and days 9 to 14, respectively. Density of CD11b molecules on milk macrophages and the ratios of phagocyte to lymphocyte percentages and polymorphonuclear cell to macrophage percentages in milk differentiated the early from the late inflammatory phase. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of bovine mammary gland macrophages and T cells in response to intramammary infusion of S. aureus was associated with an inability to culture this bacterium from milk. Identification of specific inflammatory phases of S. aureus-induced mastitis in cows may allow for the design of more efficacious treatment and control programs.     

Blood and milk cellular immune responses of mastitic non-periparturient cows inoculated with Staphylococcus aureus

Lymphocyte function and phenotype of peripheral blood and mammary gland cells were evaluated in non-periparturient cows before and at 1, 4 to 8 and 9 to 14 d after inoculation with Staphylococcus aureus, as expressed by percentage of CD3+, CD2+, and CD45R+ cells, antigen density of these markers per lymphocyte, and mitogen-induced blastogenesis. Milk bacterial counts and somatic cell counts (SCC) were also assessed. Mitogen-induced blastogenic responses were strong in blood and weak in mammary gland cells in all observations and positively correlated with the percent of CD45R+ cells. Significantly greater percentages of milk CD3+ lymphocytes and increased CD3, CD2, and CD45R antigen density per cell were observed after challenge. The blood CD3 and CD2 antigen density per lymphocyte and the milk CD2+ lymphocyte percent were negatively correlated with SCC (P ≤ 0.01). No mastitis (SCC ≤ 500 000 cells/mL) was observed in cows showing blood lymphocyte CD2 and CD3 antigen density indices ≥ 2.5 and 6, respectively. Forty-one percent of SCC values were predicted by the combined blood CD2 and milk CD3 antigen density (P ≤ 0.01). These findings support the hypotheses that mitogen-induced lymphocyte blastogenesis is not a valid test to assess mammary gland immunocompetence and that CD2 expression may facilitate immune responses by decreasing the number of T cell receptors required to achieve full activation.     

Immunohistochemical study of the inflammatory infiltrate associated with feline cutaneous squamous cell carcinomas and precancerous lesions (actinic keratosis).

The distribution of T lymphocytes (CD3+), B lymphocytes (CD79+), immunoglobulin-containing plasma cells (IgG, IgM and IgA), macrophages (Mac387+) and MHC Class II antigen was analysed in the inflammatory infiltrate associated with cutaneous squamous cell carcinomas (SCC) from 23 cats. Peri-tumoural skin (12 cases) and precancerous lesions of actinic keratosis (nine cases) were also evaluated for the expression of MHC Class II. The results revealed that an abundant inflammatory infiltrate was associated with the majority of SCC. This infiltrate was composed mainly of CD3+ T lymphocytes, B cells (CD79+) and IgG-bearing plasma cells, and the intensity of infiltration increased with the degree of invasiveness of the tumour. The number of CD3+ T cells and CD79+ cells was significantly increased in well-differentiated SCC compared with moderately differentiated tumours, whereas the number of IgM+, IgA+ plasma cells and Mac387+ macrophages was low or moderate and did not change significantly with histologic grade or invasiveness. MHC Class II antigen was expressed by infiltrating lymphocytes and macrophages, and by fibroblasts. A variable number of neoplastic cells (10% to 80%) in 10 SCC, and keratinocytes of basal layers in seven of nine cases of actinic keratosis also expressed MHC Class II, whereas keratinocytes of normal skin were always negative for this antigen. These results suggest that CD3+ T lymphocytes, CD79+ B cells and IgG-bearing plasma cells may participate in down-regulation of tumour growth, since these cell types were particularly numerous in well-differentiated and mildly invasive SCC, as well as in actinic keratosis. The expression of MHC Class II by neoplastic cells could enhance this local anti-tumour immune response.     

An outbreak of systemic granulomatous disease in cows with high milk yields

Seven of 92 lactating Holstein cows on a dairy farm developed urticaria with alopecia and decreased milk production, and three of the seven died over the course of 7 to 18 days. Pathologic examination of the three cows, including the two dead and one euthanized cow, revealed that the skin, liver, spleen, kidneys, heart, salivary glands, pancreas, adrenal glands, mammary glands, lymph nodes, and trigeminal ganglia had lymphocytic to lymphogranulomatous inflammation. Inflammation predominated by lymphocytic infiltration was prominent in the heart, pancreas, mammary glands, adrenal gland, and trigeminal ganglia. Severe granulomatous inflammation with multinucleated giant cells was present in the spleen and kidneys. These lesions and their distributions were most similar to those seen in suspected cases of citrus pulp toxicosis and hairy vetch toxicosis. The outbreak of this disease resolved with the elimination of Citrus pulp from the feed. Immunohistochemical detection of lymphocytes and macrophage markers confirmed dramatic hyperplasia of CD3-positive T lymphocytes in these lesions. This strongly suggested that a type 4 hypersensitivity reaction played a role in the development of the lesions.     

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever.

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P less than 0.001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P less than 0.001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220-) cells (P less than 0.01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8-). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.     

Effects of intra-mammary bacterial infection with coagulase negative staphylococci and stage of lactation on shedding of epithelial cells and infiltration of leukocytes into milk: comparison among cows, goats and sheep

The effects of mammary gland bacterial infection and stage of lactation on leukocyte infiltration into the mammary gland were compared among cows, goats and sheep. Animals were at two stages of lactation: mid or late. In mid-lactation animals, bacterial-free glands and coagulase negative Staphylococcus (CNS)-infected glands were compared. In late lactation only uninfected glands were studied. Of mid-lactation bacteria-free animals, goats had the highest number of leukocytes and % polymorphonuclears (PMNs), whereas sheep had the lowest and leukocytes number in cows were intermediate between sheep and goats. Based on %PMN, two cell clusters were found in sheep, which overlapped with the parallel cell clusters of cows and goats, but with a slightly higher number of leukocytes in each cell cluster. At late lactation, goats had higher values for %PMN and leukocyte numbers in comparison to cows, which had a similar cellular profile to sheep. The cellular immune response to CNS infection was similar for the three animal species, although the number of cells was different, while the basal cell level at mid-lactation and especially at the end of lactation was species specific.     

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.     

Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture

This study describes the distribution of CD4+ and CD8alpha+ T lymphocytes, B lymphocytes, macrophages, MHC class II antigens, immunoglobulin (IgG, IgM, IgA)-containing cells and of adhesion molecules belonging to the CD11/CD18 family in synovial membrane biopsies from 28 dogs with spontaneous rupture of the cranial cruciate ligament (CCL). Synovial membranes from 11 dogs without evidence of joint lesions were used as control tissues. The main cell types in synovial membranes from dogs with CCL rupture were B lymphocytes and plasma cells belonging to the IgG isotype. The severity of inflammatory cell infiltration in CCL cases was positively correlated with the expression of adhesion molecules. Double immunofluorescence labelling of frozen sections revealed that in the inflamed synovium of dogs with CCL rupture numerous dendritic cells expressing MHC class II antigen and canine CD1c were present. The findings further support the view that in the synovium of dogs with CCL rupture an immunologic response is going on in which dendritic cells are possibly involved by presenting hitherto unknown antigens to T lymphocytes.     

Expression of adhesion molecules on milk and blood lymphocytes from periparturient dairy cattle with Johne's disease

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.     

Ultrastructure and frequency of mastitis caused by ovine progressive pneumonia virus infection in sheep

Twenty-five sheep, experimentally (n = 15) or naturally (n = 6) infected with ovine progressive pneumonia virus and noninfected controls (n = 4), were evaluated for histological and ultrastructural lesions of mastitis. Histologically, nine of 15 experimentally infected sheep and all six naturally infected sheep had lympho-plasmacytic mastitis. Severity of the lesion increased with length of time after infection. Periductal lymphatic nodules were seen in five sheep experimentally infected for 2.8 years or longer and in five naturally infected sheep that were 3.7 years old or older. Ultrastructurally, responses to ovine progressive pneumonia virus were diffuse lympho-plasmacytic infiltrates in glandular interstitium, lymphocytic and occasional plasmacytic infiltrates in ductal walls and lumens, lymphoblasts surrounded by small lymphocytes in glandular interstitium, and degeneration of epithelium releasing cells and cellular debris into the lumen. Based on the prevalence of lesions, the mammary tissue was more susceptible to ovine progressive pneumonia virus than other target organs: lung, brain, and synovium. Lesions did not differ between breeds of sheep. Ovine progressive pneumonia virus was not seen in the mammary tissue but was isolated from 15 of 17 mammary glands.     

Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules

Multiparameter flow cytometry analysis and specific cluster differentiation (CD) molecules were used to determine the expression profiles of B- and T-cell antigens on lymph node preparations from 59 dogs with generalized or multisystemic lymphoma. Lymph node samples from 11 healthy dogs were labeled to validate the specificity of antibodies and to formulate guidelines for interpretation of the results obtained from lymphoma samples. In normal lymph nodes, T-lymphocytes expressing CD3, CD4, or CD8 beta represented 59+/-11%, 43+/-8%, or 16+/-5% of the total cells, whereas B-lymphocytes expressing either CD21 or surface IgM (IgM) represented 37+/-9% or 14+/-5%, respectively. Small lymphocytes could be distinguished from large lymphocytes by forward light scatter. Of the patient samples 29 different breeds were represented with Golden and Labrador retriever being the most common. The lymphoma samples segregated into three groups based on CD antigen expression. Thirty cases predominantly expressed one or more combinations of CD79a, IgM, and CD21 representing a B-cell lineage. Three B-cell cases also expressed the stem cell antigen, CD34. Sixteen cases expressed one or more combinations of CD3, CD4, and CD8 consistent with a T-cell lineage and CD3+CD4+CD8--phenotype was the most common. Thirteen cases showed a mixed expression profile for T- and B-cell antigens and in three cases CD14 was highly expressed. Clinical response was poorest for T-cell lymphomas. Leukemic states occurred in all three phenotypes; but mixed cell cases had the greatest proportion. Dual immunofluorescence staining confirmed co-expression of T-cell (CD3) and B-cell antigens (CD79a or CD21) on neoplastic lymphocytes of six mixed cell cases. In one mixed cell case, dual immunostaining identified lymphocyte populations that stained mutually exclusive for CD79a and CD3. Six mixed cell lymphomas tested by PCR showed clonality for rearranged antigen receptor. Four cases that were CD79a+CD3+ had TCRgamma chain gene rearrangements, whereas two cases that were CD3+CD8+CD21+ had Ig heavy chain rearrangement. One case expressing multiple CD molecules (CD3+CD8+CD21+CD14+) was PCR negative for both Ig and TCRgamma gene rearrangement and could not be classified into a B- or T-cell lineage. We show for the first time co-expression of B- and T-cell markers on lymphoma cells that had specific T- or B-cell gene rearrangements. These findings suggest that aberrant CD molecule expression is not an uncommon finding in canine lymphomas and is a useful diagnostic marker for malignancy.     

Immunocytochemical demonstration of lymphocyte subsets and MHC class II antigen expression in synovial membranes from dogs with rheumatoid arthritis and degenerative joint disease

The study describes the distribution of canine leucocyte antigens in synovial membrane biopsies from six dogs with canine rheumatoid arthritis (CRA) and from eight dogs with osteoarthritis (OA) secondary to spontaneous rupture of the cranial cruciate ligament (CCL) (n = 5) or patellar luxation (n = 3). Synovial membranes from five dogs without evidence of joint lesions were used as control tissues. In the subsynovium of dogs with normal joints CD5+, CD4+, CD8+ and alpha beta TCR+ lymphocytes were present only in low numbers. With monoclonal antibody (mAb) to MHC class II antigen, either none or up to 20-30% of synovial lining cells were immunoreactive. Furthermore, scattered MHCII+ stromal cells were seen in the deeper subsynovial layer. In synovial membrane biopsies from dogs with CRA numerous diffusely and perivascularly distributed CD5+ lymphocytes were found in the subsynovium. CD4+ cells outnumbered CD8+ cells and were more numerous in the perivascular areas. In all the CRA cases examined, there were markedly higher numbers of alpha beta TCR+ cells compared with gamma delta TCR+ cells. With mAb to CD21, low numbers of immunoreactive lymphocytes were demonstrated. In all the CRA cases, a marked increase of MHC class II antigen expression was noted. In the majority of samples, 50% or more than 90% of the synovial lining cells were strongly MHC class II+. Throughout the subsynovial layer there were numerous MHC class II+ cells and included those with dendritic morphology and inflammatory mononuclear cells. Furthermore, marked perivascular immunoreactivity for MHC class II antigen was found. In biopsies from dogs with OA, there were markedly lower numbers of subsynovial CD5+, CD4+ and CD8+ lymphocytes. T-cells were mainly diffusely distributed. In three of the eight OA dogs examined, there was an increased percentage of synovial lining cells expressing MHC class II. The majority of OA cases had subsynovial major histocompatibility complex (MHC) class II+ cells with a dendritic morphology.     

Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score,somatic cell count,and milk mononuclear cell populations in Holstein cows with <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> subclinical mastitis

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 μg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

Expression of lymphocyte homing and adhesion molecules during intramammary infection of cows with Serratiamarcescens or Streptococcusuberis: correlation with bacterial colonization and clinical signs

We wished to determine the expression of trafficking/adhesion molecules on the surface of lymphocytes isolated from infected mammary glands of cows challenged with either Serratia marcescens or Staphylococcus uberis. Healthy Holstein cows in mid lactation were infected by intramammary infusion with S. marcescens or S. uberis. Following infection, milk samples were collected at various time points. Body temperatures of the cows were taken, and milk was analyzed for colony forming units (CFU) of bacteria and somatic cell counts (SCC). Leukocytes were isolated from the milk and analyzed by flow cytometry. Percentages and types of lymphocytes were determined as well as expression of CD62L, CD11a, LPAM-1 and CD44 on these cells. We found that the percentage of lymphocytes expressing either CD62L or CD11a showed a marked increase 12 h post infection (PI) with S. marcescens that was not seen in cows infected with S. uberis. Conversely, the percentage of lymphocytes expressing CD44 increased in cows infected with S. uberis at 12 h PI, but the increase was not seen in cows infected with S. marcescens. Expression of LPAM-1 was low at all time points in both groups of cows. Body temperatures became elevated in both groups of cows, peaking at 24 h PI in S. marcescens-infected cows and dropping thereafter. In contrast, temperatures of S. uberis-infected cows continued to rise and were still elevated 96 h PI. CFU of bacteria isolated from mammary glands of S. marcescens-infected cows dropped precipitously 24 h PI but continued at high levels in S. uberis-infected cows. SCC began falling in S. marcescens-infected cows 48 h PI but continued to increase in S. uberis-infected cows. Thus, a greater percentage of lymphocytes in milk had a phenotype consistent with recruitment from the peripheral pool following infection with S. marcescens than was seen following infection with S. uberis. Concurrent with the increases seen in percentages of this lymphocyte phenotype, clinical signs lessened in the S. marcescens-infected cows.     

Colonic lymphocyte and plasma cell populations in dogs with lymphocytic-plasmacytic colitis

OBJECTIVES: To quantitate immunoglobulin-containing cells (IgA, IgG, and IgM) and CD3+ T cells in colonic biopsy specimens obtained from dogs with lymphocytic-plasmacytic colitis (LPC), and to compare lymphocyte and plasma cell populations in dogs with LPC with those in healthy dogs. ANIMALS: 10 healthy dogs and 11 dogs with LPC. PROCEDURE: Colonic mucosal specimens obtained from healthy dogs and dogs with LPC were stained specifically for IgA-, IgG-, and IgM-containing cells and CD3+ T cells by use of immunoperoxidase techniques. Morphometric analyses were done to quantitate lymphocytes and plasma cells in standardized areas of colonic mucosa. Data analyses allowed determination of mean cell numbers in each dog group, and comparison of mean numbers of lymphocytes and plasma cells between dog groups. RESULTS: CD3+ T cells predominated in healthy dogs, whereas CD3+ T cells and IgA-containing cells were most numerous in dogs with LPC. In both dog groups, the IgG- and IgM-containing cells were considerably less numerous than the other 2 cell types. Comparison of cell populations between dog groups indicated that IgA- and IgG-containing cells and CD3+ T cells were significantly more numerous in the colonic mucosa of dogs with LPC. CONCLUSIONS: Dogs with LPC have significantly increased numbers of IgA- and IgG-containing cells and CD3+ T cells. These lymphocyte and plasma cell distributions indicate similarities to and differences from such distributions in human beings with inflammatory bowel disease. Results provide a basis for future correlation between histologic stage of disease activity and immunologic findings in dogs with LPC.     

Lymphocytes and T lymphocyte subsets are regionally distributed in the female goat reproductive tract: influence of the stage of the oestrous cycle

The reproductive tract of the female is a part of the mucosal system which protects from pathogens invasion. We have analysed the presence and distribution of total lymphocytes, plasma cells (antibody secreting B cells) and T lymphocytes subsets in the reproductive tract of the female goat. The influence of the oestrous cycle on the densities of lymphocytes and plasma cells of the cervix and uterus horn was evaluated in sections prepared for conventional histology. Immunocytochemistry was used for the study of lymphocyte subsets by confocal microscopy and immunoperoxidase techniques. Present results show that the reproductive tract of the goat is a site rich in lymphocytes. These cells were found mingled with the epithelial cells of the endometrium and distributed throughout the stroma. Lymphocyte aggregates were observed in the stroma. Lymphocyte but not plasma cell number changed depending on the reproductive stage of the goats. The impact of the hormonal environment was different for the cervix and uterine horn. Immunocytochemistry studies evidenced the presence of cells displaying immunoreactivity for both CD 4+ and CD 8+ antibodies in the epithelial layer and stroma of the cervix and uterine horn. These cells were more numerous in the cervix and were also found infiltrating the luminal epithelia of endometrial glands. Overall, our results indicate that lymphocyte distribution is different in the cervix and the horn, and is influenced by the stage of the reproductive cycle. In summary, CD 4+ and CD 8+ T lymphocytes subsets could be found in the endometrium of both the cervix and uterine horn of the goat reproductive tract.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

Studies on the modulation of leucocyte subpopulations and immunoglobulins following intramammary infusion of beta 1,3-glucan into the bovine udder during the dry period

Inflammatory and immunological reactions after intramammary infusion of beta 1,3-glucan were studied during the steady dry period and involution phase of the bovine udder. The effects of a single intramammary infusion of two different doses (100 and 200 mg) of beta 1,3-glucan were evaluated during the steady dry period. In a second study, the effects of beta 1,3-glucan at drying off were studied by using two treatment regimens; a single infusion at drying off, compared with two infusions of the compound, at drying off and again 2 weeks later. Total and differential leucocyte counts were measured in both blood and udder secretions. Additionally, the expression of receptors for CD14 and MHC class II on leucocytes, and the expression of receptors for CD4, CD8, WC1, IL2R and B-cells on lymphocytes was measured in mammary secretions by flow cytometric analyses. The concentrations of immunoglobulins in udder secretions were measured by radial immunodiffusion. The results showed that a single intramammary infusion of beta 1,3-glucan during the steady dry period causes transient enhancement of some aspects of the inflammatory and immune responses. The increases in somatic cell counts, numbers of monocytes/macrophages, and in proportions of CD14+ and MHC class II+ leucocytes in udder secretions were dose-dependent. Infusion of beta 1,3-glucan also slightly increased the proportion of CD4+ lymphocytes and the concentrations of IgG1 and IgG2 in dry secretions. Infusion of beta 1,3-glucan at drying off seemed to accelerate the involution process through an increase in somatic cells, particularly in the numbers of macrophages, in mammary secretions. The numbers of lymphocytes and polymorphonuclear leucocytes, the proportions of IL2R+ lymphocytes, the proportions of CD14+ or MHC class II+ leucocytes and the concentrations of IgG1 and IgG2 also increased in comparison with untreated controls. Moreover, a second infusion of beta 1,3-glucan tended to prolong this response, indicating that this might be an effective means of enhancing the mammary defence against udder infections closer to calving. In conclusion, the results indicate the intramammary infusion of beta 1,3-glucan could be used to enhance the defence mechanisms of the bovine udder against infections, especially during early involution.