Analysis of blood leukocytes in a naturally occurring immunodeficiency of pigs shows the defect is localized to B and T cells.

Severe combined immunodeficiency (SCID) is the result of a set of inherited genetic defects which render components of the immune response nonfunctional. In Arabian horses, Jack Russell terriers, and mice, the disorder is a consequence of the absence of T and B lymphocytes, while natural killer (NK) cell and other leukocyte populations remain intact. Preliminary analysis of a naturally acquired form of inherited SCID in a line of pigs showed several defects in the architecture and composition of secondary lymphoid organs. In this study, a quantitative assessment of lymphocyte populations in affected and normal littermates showed depleted T or B lymphocyte populations in affected pigs; however, NK cells and neutrophils were present in numbers comparable to unaffected littermates. The results indicate that the immune defect in pigs shares the same features as other SCID-affected species.

Association of cartilage defects, and other MRI findings with pain and function in individuals with mild-moderate radiographic hip osteoarthritis and controls.

OBJECTIVE: To evaluate the relationship of hip radiographic osteoarthritis (ROA) and MRI findings of cartilage lesions, labral tears, bone marrow edema-like lesions (BMELs) and subchondral cysts with self-reported and physical function. DESIGN: Eighty five subjects were classified as controls (n = 55, Kellgren-Lawrence (KL) 0, 1) or having mild-moderate ROA (n = 30, KL 2, 3). T2 weighted MRI images at 3-T were graded for presence of cartilage lesions, labral tears, BMELs and subchondral cysts. Posterior wall sign, cross-over sign, center-edge angle and alpha angle were also recorded. Function was assessed using Hip dysfunction and Osteoarthritis Outcome Score (HOOS), Timed-Up and Go (TUG) test and Y-Balance Test (YBT). Analysis compared function between subjects with and without ROA and those with and without femoral or acetabular cartilage lesions, adjusted for age. Non-parametric correlations were used to assess the relationship between radiographic scores, MRI scores and function. RESULTS: Subjects with acetabular cartilage lesions had worse HOOS (Difference = 5-10%, P = 0.036-0.004), but not TUG or YBT, scores. Acetabular cartilage lesions, BMELs and subchondral cysts were associated with worse HOOS scores (ρ = 0.23-0.37, P = 0.041-0.001). Differences in function between subjects with and without ROA or femoral cartilage lesions were not significant. Other radiologic findings were not associated with function. CONCLUSIONS: Acetabular cartilage defects, but not femoral cartilage defects or ROA, were associated with greater self-reported pain and disability. BMELs and subchondral cysts were related to greater hip related self-reported pain and disability. None of the radiographic or MRI features was related to physical function.

Preliminary findings of a previously unrecognized porcine primary immunodeficiency disorder.

Weaned pigs from a line bred for increased feed efficiency were enrolled in a study of the role of host genes in the response to infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Four of the pigs were euthanatized early in the study due to weight loss with illness and poor body condition; 2 pigs before PRRSV infection and the other 2 pigs approximately 2 weeks after virus inoculation. The 2 inoculated pigs failed to produce PRRSV-specific antibodies. Gross findings included pneumonia, absence of a detectable thymus, and small secondary lymphoid tissues. Histologically, lymph nodes, spleen, tonsils, and Peyer's patches were sparsely cellular with decreased to absent T and B lymphocytes.

Factors affecting the permissiveness of porcine alveolar macrophages for porcine reproductive and respiratory syndrome virus.

Alveolar macrophages from PRRSV-infected and naïve pigs were placed into culture and infected with PRRSV laboratory strain SD-23983. Permissiveness increased with time in culture, and macrophages from infected pigs could be superinfected. Addition of actinomycin D, an inhibitor of mRNA synthesis, blocked infection. Interferon-gamma reduced infection in cultures, while the addition of tumor necrosis factor-alpha or interleukin (IL)-10 did not affect permissiveness. IL-4 produced a marginal increase in the percentage of infected cells, but without a detectable increase in virus yield. These results suggest that the PRRSV-permissive population of cells in culture arises from a non-permissive precursor population and depends on new mRNA synthesis.

Response of T lymphocytes from previously infected calves to recombinant Cryptosporidium parvum P23 vaccine antigen.

We had previously demonstrated that a Type-1-like immune response involving interferon-gamma expression in lamina propria lymphocytes accompanied by IgG2 subclass fecal antibodies to Cryptosporidium parvum p23 emerged in gut mucosa of calves recovering from cryptosporidiosis. Because a recombinant p23 had been shown to protect calves from cryptosporidiosis when administered as a vaccine antigen to late gestation cattle, this study was undertaken to determine if the same vaccine antigen could induce a Type-1-like, in vitro response by T cells from calves that had recovered from C. parvum infection. We isolated peripheral blood mononuclear cells from calves that had been previously infected with C. parvum oocysts and incubated them in the presence or absence of the recombinant C. parvum p23 vaccine antigen. We used flow cytometry to simultaneously detect cells in cell cycle and identify the T cell subset containing cycling cells. We also used flow cytometry to identify interferon-gamma positive cells and 2-dimensional gel electrophoresis to profile proteins made by PBMC stimulated with the recombinant p23 vaccine antigen. The results demonstrated that CD4+ T lymphocytes proliferated and that interferon-gamma was synthesized by a subset of stimulated cells. Two-dimensional gel electrophoresis revealed the presence of several cytoplasmic proteins in a size range of approximately 25-80 kDa that were detected in p23-stimulated, but not in unstimulated, cytoplasmic samples. Together, the results show that the recombinant p23 vaccine antigen can stimulate a Type-1-like immune response by T cells from calves that have recovered from C. parvum infection.

Modulation of bovine lymphocyte response by salivary gland extracts of the stable fly, Stomoxys calcitrans (Diptera: Muscidae).

The effect of salivary gland extract of the stable fly, Stomoxys calcitrans (L), on bovine lymphocyte proliferation was determined, and antibody reactivity to salivary gland proteins was characterized in cattle exposed to stable flies. Salivary glands were dissected from male and female flies (4-8 d after eclosion), and protein extracts were made by freeze-thaw cycles. Salivary gland extract (SGE, 1 and 5 microg) significantly inhibited mitogen-driven proliferation of bovine lymphocytes, compared with 1 and 5 microg of identically prepared midgut extract (ANOVA, P < 0.05). Phytohemagglutinin A (PHA) stimulated lymphocyte responses were suppressed by 61.7 and 79.5% (mean values) with 1 and 5 microg of SCE, whereas concanvalin A (Con A) stimulated responses were suppressed by 62.9 and 77.1% (1 and 5 microg). In contrast, midgut extract (1 and 5 microg) minimally suppressed PHA (12.7% +/- 12.6 and 18.7% +/- 15.5) and Con A-driven responses (13.8% +/- 20.5 and 24.6% +/- 14.9), respectively. Viability studies using propidium iodide and flow cytometry demonstrated that SGE was not cytotoxic. Two-color immunofluorescence studies identified T and B lymphocytes as the nonviable cells in the cultures. Western blot analysis of serum collected from five dairy cows during periods of low and high fly exposure identified an immunodominant 27 kDa protein among the salivary gland proteins. These results indicate that exposure of cattle to stable fly saliva during blood feeding results in an antibody response to salivary proteins and that the saliva has a potential to modulate T lymphocyte function.

Evidence for the emergence of a type-1-like immune response in intestinal mucosa of calves recovering from cryptosporidiosis.

This study was undertaken to characterize the mucosal response to Cryptosporidium parvum in infected calves that had recovered from diarrhea. Flow cytometric surface phenotypes of lamina propria lymphocyte (LPL) suspensions from infected calves and age-matched controls revealed the presence of a significantly larger proportion of CD25+ LPL in infected calves than in controls. Freshly isolated LPL from infected calves expressed more iNOS and interferon (IFN)-gamma than did controls. Infected calves excreted IgG1 and IgG2 isotype antibodies to C. parvum p23 by the end of the experiment. Moreover, immunohistochemistry of ileal sections revealed the presence of IgG1+ and IgG2+ B lymphocytes in the villi and IgG1+ but not IgG2+ B lymphocytes in continuous Peyer's patch nodules. These data are consistent with the emergence of a type-1-like mucosal immune response in terminal ileal mucosa as calves recover from cryptosporidiosis.

Excretion patterns of mucosally delivered antibodies to p23 in Cryptosporidium parvum infected calves.

Fecal samples obtained at intervals from six calves with acute cryptosporidiosis contained antibodies of multiple isotypes to p23. IgM-, IgA-, and IgG(1)-isotype anti-p23 appeared before IgG(2)-isotype antibodies. All anti-p23 antibodies had declined by 2 months after infection. One calf that failed to shed oocysts following initial exposure developed IgG(1)-isotype anti-p23 antibodies. One calf that died following exposure to Cryptosporidium parvum oocysts lacked detectable anti-p23 antibodies. Re-inoculation with C. parvum resulted in a brief, marked recall response to p23.

Detection of mucosally delivered antibody to Cryptosporidium parvum p23 in infected calves.

We have developed an assay to detect mucosally delivered antibody to Cryptosporidium parvum sporozoite antigens. We absorbed a recombinant 23-kD sporozoite protein to polystyrene microspheres, and used flow cytometry to detect, titer, and determine the isotype of antibody to p23 that was shed in the feces of experimentally infected calves. Noninoculated calves have low levels of mucosal antibody to p23, with IgG1 as the predominant isotype. Antibody titers rise in inoculated calves as the animals recover from cryptosporidiosis. A calf that was naturally protected from cryptosporidiosis had mucosal IgM and IgG1 isotype anti-p23 antibodies prior to challenge with C. parvum oocysts. Ten days after challenge, the calf had high titers of IgM, IgA, IgG1, and IgG2 anti-p23 antibodies. Together, the data show that this method can be used to assess mucosally delivered antibody to C. parvum.

Cryptosporidium parvum and mucosal immunity in neonatal cattle.

Cryptosporidium parvum is an important zoonotic protozoan pathogen that causes acute infection and self-limiting gastrointestinal disease in neonatal calves. There are currently no consistently effective antimicrobials available to control cryptosporidiosis. Therefore, immunotherapeutic and vaccination protocols offer the greatest potential for long-term control of the disease. In order to devise effective control measures, it is important to better define mucosal immunity to C. parvum in young calves. This review summarizes the information that has accumulated over the last decade which helps to define the intestinal mucosal immune system in neonatal calves, and the events that occur in the intestinal mucosa after infection by C. parvum.

Accumulation of mucosal T lymphocytes around epithelial cells after in vitro infection with Cryptosporidium parvum.

We had previously shown that ileal intraepithelial lymphocytes isolated from calves with cryptosporidiosis include significantly increased numbers of CD8+ T lymphocytes and activated CD4+ cells. These increases could result from redistribution of resident mucosal lymphocytes or from homing of peripheral T cells to ileal mucosa. To determine whether resident mucosal lymphocytes can redistribute to Cryptosporidium parvum-infected epithelium, oocysts were inoculated in vitro onto ileum explants taken from 1-2-wk-old noninfected calves. After 24 hr of incubation, the explants were collected and frozen in liquid nitrogen. Immunohistochemical analysis of T-lymphocyte subpopulations was performed on sections, and labeled lymphocytes adjacent to villous epithelial cells were counted. Compared with uninoculated explants, there was a statistically significant increase in the number of CD8+ T lymphocytes per 100 epithelial cells in oocyst-inoculated tissue. In addition, there were increased numbers of CD4+ T cells and activated (CD25+) lymphocytes adjacent to C. parvum-infected epithelium. These results show that resident mucosal T lymphocytes can accumulate at the epithelium during C. parvum infection.

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.

The role of IL-10 in iNOS and cytokine mRNA expression during in vitro differentiation of bovine mononuclear phagocytes.

In the study reported here, we used RT-PCR with primers specific for interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide synthase (iNOS) to assess the cytokine mRNA expression associated with bovine blood monocytes during their differentiation to macrophages cultured on plastic (1 week). In addition, we used RT-PCR to assess the contribution of gammadelta T cells as a source of interferon-gamma (IFN-gamma), the induction signal for iNOS. Further, we evaluated cytocentrifuge preparations from the cultures for the production of IL-10 using specific antibody. We previously demonstrated that iNOS can be induced in cultured bovine monocytes in response to IFN-gamma and TNF-alpha but lose this capability in a short period of time. However, we demonstrate here that iNOS induction from monocytes cultured with IFN-gamma secreting gammadelta T cells is prolonged, suggesting that this source of IFN-gamma primes the monocytes before exogenous stimulation. Based on mRNA expression, placement of monocytes in culture resulted in activation, followed by quiescence. By 6 days in culture, the iNOS message was reduced below the basal level. In addition, the TNF-alpha message was substantially reduced, and IL-1 and IL-6 messages were reduced below detectable levels. This correlated with an increase in IL-10 message. Downregulation of these same cytokine messages as well as IFN-gamma message occurred within a 20-h period when IL-10 was added exogenously to cultures of total leukocytes. At the same time, there was an increase in the number of IL-10-positive cells and an increase in the intensity of anti-IL-10 staining within adherent cells. These results provide evidence for IL-10 regulation of some bovine mononuclear phagocyte effector functions.

Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum.

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.

Identification of two distinct subsets of bovine gamma delta T cells with unique cell surface phenotype and tissue distribution.

We describe the characterization of two subsets of bovine gamma delta T cells having distinct cell surface phenotype and tissue distribution. One population expresses the previously described 215,000 MW WC1 antigen and is negative for the cell-surface differentiation antigens CD2, CD4, and CD8. The second population expresses CD2 and CD8 but not WC1 and appears to have a T-cell receptor (TCR) rearrangement distinct from that of the WC1+ population. The WC1- population is found in large numbers in spleen and intestine. In addition, this subset is not recognized by a number of monoclonal antibodies (mAbs) specific for TCR families that are well represented in the WC1+ population. The results indicate that the gamma delta T-cell population in cattle is considerably larger than previously described and that this population can be subdivided into two distinct subsets based on cell-surface phenotype and tissue distribution.

Assessment of bovine mononuclear phagocytes and neutrophils for induced L-arginine-dependent nitric oxide production.

Microbicidal activity of reactive oxygen intermediates and reactive nitrogen intermediates has been described from both murine and human cytokine activated macrophages. An L-arginine-dependent pathway of nitric oxide generation has recently been described from bovine bone marrow-derived and monocyte-derived macrophages in response to a phagocytic stimulus. We have investigated the induction and release of both reactive oxygen intermediates and reactive nitrogen intermediates from bovine neutrophils, and blood and spleen mononuclear phagocytes in response to either a phagocytic or cytokine stimulus. Mononuclear phagocytes were poor producers of hydrogen peroxide (a measure of reactive oxygen intermediate production) under conditions that readily caused release by neutrophils. In contrast, nitrite, as a measure of nitric oxide production, could not be induced from neutrophils under any stimulation conditions, while mononuclear phagocytes responded to both a phagocytic stimulus and cytokines with the induction of nitric oxide synthase message and production of nitric oxide. There appeared to be two populations of monocytes that differed both in their adherent characteristics and their level of cytokine-induced nitric oxide production. Both populations stained with a single monoclonal antibody. However, the population that had not adhered to plastic within 3 h responded to cytokine stimulation, producing up to 3 times more nitric oxide on a per cell basis than the readily adherent population. Cytokine induction required the presence of interferon-gamma and either tumor necrosis factor-alpha or lipopolysaccharide. L-arginine dependence was demonstrated by inhibition with an L-arginine analog and restoration with addition of excess L-arginine.

Effect on intraerythrocytic Anaplasma marginale of soluble factors from infected calf blood mononuclear cells.

Blood mononuclear cells (lymphocytes and monocytes) were isolated from infected calves during in vivo control of acute anaplasmosis and cultured with Anaplasma marginale organisms. Supernatants from the cultures reduced the proportion of erythrocytes containing viable A. marginale in vitro, indicating that an antibody-independent mechanism of rickettsemia control might occur during acute anaplasmosis.

Analysis of monoclonal antibodies specific for the gamma delta TcR.

gamma delta T cells in ruminants can be subdivided in two or more subpopulations on the basis of the expression of surface antigen WC1, which can exist in different isoforms. In this study, 18 monoclonal antibodies (mAbs) submitted to the Third International Workshop that were predicted to react with gamma delta TcR molecules were analysed and expression of their antigens was investigated on the different gamma delta T cell subpopulations. A set of control mAbs positive for TcR1 (86D), BoCD3 (MM1A), WC1 (B7A1, BAQ4A, CACTB32A, and BAQ89A) was included for comparative studies. Previous investigations demonstrated eight of the mAbs immunoprecipitated peptides with apparent M(r)s of 37 and/or 47 kDa, indicating they recognized determinants on the T cell receptor, TcR1. Two color flow cytometric analyses in the present study demonstrated the mAbs formed three groups; group 1, a set of mAbs that recognize TcR1 determinants expressed on all gamma delta T cells and groups 2 and 3, sets of mAbs that recognize TcR1 determinants on some gamma delta T cells: TcR1-N6 and TcR1-N7 respectively. mAbs from the latter groups define families of TcR1 molecules that express either one or both of the determinants. These antigenically distinct forms of TcR1 are expressed in equal proportion on the two gamma delta T cell populations that express one of the mutually exclusive isoforms of WC1, WC1-N3 and WC1-N4. The data indicate usage of the mAb-defined families of the gamma delta TcR is primarily restricted to the WC1+ subpopulation of gamma delta T cells. However, a small subpopulation of CD2+, WC1- gamma delta T cells expresses a form of TcR1 positive for the determinant TcR1-N6.

Reactive oxygen and nitrogen intermediates and products from polyamine degradation are Babesiacidal in vitro.

Products released from activated macrophages have been demonstrated to have microbicidal activity against a variety of microorganisms. Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) have been shown to affect the induction of degenerate (crisis) forms of Plasmodium spp. Polyamines are degraded into acrolein which has also been shown to be toxic to Plasmodium spp. We have investigated the possibility that these products act similarly with Babesia bovis. Crisis forms of B. bovis developed in erythrocyte cultures after the introduction of supernatants containing ROI, RNI, and acrolein. Xanthine degradation by xanthine oxidase leads to the formation of superoxide anion, hydrogen peroxide, and hydroxyl radicals. The degradation in the presence of B. bovis was toxic to the parasite. The toxicity was partially reversed by the addition of the ROI scavenger catalase. However, H2O2 added directly had little effect, suggesting a role for the other ROI products. Spermine degradation by polyamine oxidase and direct addition of acrolein was toxic in a dose-dependent manner. Finally, spontaneous generation of nitric oxide from sodium nitroprusside or S-nitroso-N-acetyl-penicillamine was also toxic in a dose-dependent manner. These data lead us to suggest a role for activated macrophages in the primary immune response against B. bovis.