Lymphocyte Stimulation and Leucocyte Migration Inhibition as Diagnostic Tools for the Detection of Mycobacterium Avium Infection in Cattle

The tuberculin skin test is the conventional method of detecting infections with mycobacteria in animals. A positive reaction is considered to reflect cell-mediated immunity (CMI). CMI against mycobacteria can be studied by in vitro systems using suspensions of blood lymphocytes or leucocytes. The reactivity of these cells to different antigens can be measured in the lymphocyte stimulation (LS) (Muscoplat et al 1975, Bergman 1976, Johnson & Morein 1976), or leucocyte migration inhibition (LMI) (Aalund 1970, Clausen 1973) tests.     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

Protective efficacy of capsule extracts of Haemophilus pleuropneumoniae in pigs and mice

Capsular extracts of Haemophilus pleuropneumoniae, Serotype 1, were mixed with AL(OH)3 gel (3 parts extract + 1 part Al(OH)3) and used as vaccines in pigs and mice. Four preparations were tested in Experiment I: NaCl and Cetavlon (hexadecyltrimethyl ammonium bromide) extracts of both low in vitro passage (LP) and high in vitro passage (HP) culture, respectively. Four pigs vaccinated with the NaCl extract of the LP strain survived, whereas one of four from each of the remaining vaccine groups and five of six from the control group died. All vaccines induced complement-fixing antibodies. No apparent boosting of titres occurred as a result of challenge with live bacteria. Mice were vaccinated in Experiment II with NaCl and Cetavlon extracts of the LP strain. Both were protective, although the Cetavlon vaccine appeared more efficacious than the NaCl extract. The use of Al(OH)3 adjuvant improved the efficacy of the NaCl vaccine in mice. In Experiment III six gnotobiotic pigs were vaccinated with a combined NaCl and Cetavlon vaccine and seven animals were given placebo. In Experiment IV seven specific pathogen-free (SPF) pigs were given the combined vaccine and eight pigs received placebo treatment. Both of these experiments indicated that the extract vaccines did not completely protect but reduced the mortality in pigs challenged with homologous virulent H. pleuropneumoniae bacteria. The results indicate that capsular antigens of H. pleuropneumoniae have some protective immunogenic efficacy in pigs and mice.     

Predicting resistance of sheep to Haemonchus contortus infections

In vitro cultures of lymphocytes from worm-free sheep responded to larval and adult H. contortus antigens by undergoing blast transformation. The level of response varied considerably between animals but was heritable and positively correlated with resistance to subsequent primary, secondary and trickle infection by the parasite, but not with age of the sheep older than 5 months. Lymphocyte responses to phytohaemagglutinin and H. contortus antigens were not present at birth in lambs but developed during the first 5 months in the absence of infection. The proportion of the infecting dose of larvae which was inhibited at the early fourth stage correlated with age of the sheep and with the increase in lymphocyte response to H. contortus antigens during primary infections with H. contortus. Sheep which were most susceptible to infection had the lowest lymphocyte responses to H. contortus antigens but had the highest rate of weight gain during infection. It is suggested that man has selected the most productive animals at the expense of resistance to H. contortus. The definition of genetically controlled markers on lymphocytes is seen as a means by which disease resistant strains of animals may be selected.     

Cellular immunity to canine mammary tumor cells demonstrated by the leucocyte migration technique.

Cellular immunity to canine mammary tumor cells was studied by means of the leucocyte migration technique (LMT). Intact tumor cells, separated either by enzymatical or mechanical disruption, were used as antigen, and efforts were made to cultivate tumor cells in vitro. Fifteen female tumorous dogs were studied, and 12 non-tumorous mainly male dags were used as controls. Leucocytes from tumor-bearing females were mixed with own autologous or foreign homologous tumor cells, and control leucocytes were presented with cells from the same source. In addition, leucocytes from tumorous animals and controls were mixed.Animal group A comprised 8 tumor-bearing females. In this group mixtures of different cell numbers and different tumor cell/leucocyte ratios were tried. Animal group B comprised 7 tumor-bearing females, and 40 × 10⁶ leucocytes from these were mixed with 2 × 10⁶ antigencells, antigen-cell/leucocyte ratio 0.05. A great number of tumor cells (tumor cell/leucocyte ratio > 0.05) caused strong non-specific inhibition of leucocyte migration, but in spite of marked inhibition (< 61%) in the homologous system in animal group A, inhibition in the autologous system was found to be stronger (72.2–92.3%). In animal group B, dogs presented with own tumor cells showed marked inhibition (23.7–90.1%), while the controls showed a migration inhibition below 20%. Mixtures of homologous leucocytes showed inhibition of the same order as mixtures of control leucocytes and tumor cells. Thus evidence of cellular immunity against own canine mammary tumor cells was obtained. It proved difficult to cultivate the tumor cells for more than 2–3 passages. Some evidence of antigenic cross reactivity was obtained between 2 adenocarcinomas. Enzymatical separation of tumor cells did not seem to alter antigenic characteristics of the cell surface. Mechanical separation, however, proved to be simpler, more rapid and yielded cell suspensions largely free of debris, and is therefore recommended for further work.     

Program of vaccination and antibiotic treatment to control polyserositis caused by Haemophilus parasuis under field conditions

The present study investigated the effects of vaccinating sows and piglets or piglets alone against Haemophilus parasuis on the prevalence of H. parasuis in nasal swabs, on the humoral and cellular immune responses, and on the production parameters of piglets at 3 Korean farms with a clinical history of polyserositis caused by H. parasuis. Piglets born to vaccinated or non-vaccinated sows were subdivided into 3 groups: vaccinated sows and vaccinated pigs (VS-VP), non-vaccinated sows and vaccinated pigs (NVS-VP), and non-vaccinated sows and non-vaccinated pigs (NVS-NVP). The proportion of piglets with positive nasal swabs was significantly lower (P < 0.05) in the vaccinated animals (VS-VP and NVS-VP groups) than in the non-vaccinated animals (NVS-NVP group) at 35 and 60 d of age at the 3 farms. The overall growth performance (from 7 to 60 d of age) of the vaccinated piglets was significantly better (P < 0.05) than that of the non-vaccinated piglets at the 3 farms. Piglets in the VS-VP group had significantly higher levels (P < 0.05) of H. parasuis-specific IgG antibodies, lymphocyte proliferation, and interferon-γ-secreting cells than piglets in the NVS-VP and NVS-NVP groups on days 1, 7, 21, 35, and 60 after birth at the 3 farms.     

Experimental immunization against respiratory disease due to equid herpesvirus 1 infection (rhinopneumonitis) using formalin-inactivated virus with various adjuvants

The efficacy and safety of four adjuvants, viz, alhydrogel, adjuvant 65, levamisole and killed Corynebacterium parvum were compared with Freund's complete adjuvant (FCA) for immunizing foals and yearlings with formalin inactivated, partially purified equid herpesvirus 1 (EHV-1) antigen.The levels of antibody in serum and nasal secretions and the degree of lymphocyte stimulation (LS) induced by inactivated EHV-1 antigen with FCA were higher than those following infection with a virulent strain. Levamisole and C. parvum failed to augment the antibody response to inactivated EHV-1 antigen or to induce specific lymphocyte stimulation. Adjuvant 65 and alhydrogel, although less effective than FCA, both produced good humoral and LS responses. Alhydrogel proved the most satisfactory adjuvant as FCA produced unacceptable local reactions, and adjuvant 65 was difficult to administer.Neutralizing antibody induced by immunization with inactivated RAC-H virus (subtype 1) showed remarkable strain specificity and failed to cross-react with H45 virus (subtype 2).The duration of virus excretion following intranasal challenge was reduced in immunised animals but clinical responses still occurred in some vaccinated animals when high challenge doses of virus (10^9.4TCD₅₀) were used.     

Direct and indirect lymphocyte participation in the immunity and immunopathology of tropical canine pancytopenia—A review

Erlichia canis, a rickettsial pathogen which infects monocytes, induces generalized lymphocyte activation. Activated T lymphocytes differentiate into effector cells capable of destroying infected and uninfected monocytes and platelets. Activated B lymphocytes differentiate into plasma cells associated with plasmocytosis, hypergammaglobulinemia with high levels of specific antibody and a platelet migration inhibition factor. These effector mechanisms, aimed at parasite destruction, contribute to the pathogenesis of acute tropical canine pancytopenia and do not completely eliminate the etiologic agent. A carrier state of ‘infection-immunity’ exists between the immunocompetent host and virulent parasite. E. canis has evolved mechanisms of ‘tolerant-symbiosis’ with hostile macrophages and other effector cells in the immune host.     

Effect of infections with swine fever virus on immune functions II. Lymphocyte response to mitogens and enumeration of lymphocyte subpopulations

Peripheral blood and spleen lymphocytes from pigs infected with a low-virulent strain of swine fever virus (SFV) were transiently hyporesponsive to the mitogenic action of PHA, PWM and Con A. The mitogenic reactivity of lymphocytes from lymph nodes from such pigs appeared to be enhanced rather than depressed at that time. In addition, hyperresponsiveness of peripheral blood lymphocytes (PBL) to these mitogens occurred in some pigs.PBL from pigs lethally infected with virulent SFV showed a persistent depression of the response to these mitogens, whereas lymphocytes from lymph nodes had a high responding capacity.A lymphocyte response to SFV antigens could not be demonstrated in infected pigs.These SFV infections did not markedly affect the percentage of lymphocytes in the blood and most lymphoid organs rosetting with sheep red blood cells. On the other hand, surface immunoglobulin-bearing lymphocytes were markedly increased in lymph nodes from pigs exposed to virulent SFV. The sum of both lymphocyte subpopulations in the lymph nodes from these pigs often considerably exceeded the 100% value, which strongly suggests the presence of cells bearing both surface immunoglobulin and receptors for dextran-treated sheep red blood cells.Possible correlations between these findings are discussed. The results suggest that infections with SFV induce systemic alterations in the process of lymphocyte recirculation in the pig.     

Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes

The efficacy of an adjuvanted bovine rotavirus vaccine in pregnant cattle (15 heifers and 2 cows) was studied. Each of 4 animals was inoculated IM at 8, 5, and 2 weeks before parturition with a water-in-oil emulsion containing live purified bovine rotavirus, mineral oil, and a mannide oleate compound. Four other animals were treated identically, except that muramyl dipeptide was added to the virus preparation. Five additional animals were inoculated orally at the same time intervals with adjuvant-free viral suspension, and 4 other pregnant animals inoculated only with buffer served as uninoculated controls. Kinetic studies of the specific immune responses were determined by quantification of the rotavirus-neutralizing antibodies and by a rotavirus lymphocyte stimulation test in vitro. Results showed that only the emulsions induced marked enhancement of rotavirus antibody titers in the serum, colostrum, and milk of inoculated cows. Colostral and milk lymphocytes isolated from these cows had a positive in vitro proliferative response to rotavirus stimulation, which lasted at least 21 days after parturition. The values of the stimulation index obtained with the colostral/milk lymphocytes were higher than those of the blood lymphocytes, reflecting increased lymphocyte activity in the colostrum/milk. However, addition of muramyl dipeptide to the emulsion preparation did not exert any potentiating effect on the immune response to rotavirus. Calves fed for the first 5 days after birth with a rotavirus-immune cell-free colostrum supplement were protected from a rotavirus challenge exposure on the third day after birth. Virus was not detectable in their feces.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro stimulation of pig lymphocytes after infection and vaccination with Aujeszky's disease virus

Mitogenic and antigenic lymphocyte stimulation were examined in Aujeszky's disease virus (ADV) infected pigs and in pigs vaccinated with modified live ADV. Neither infection nor vaccination had any effect on lymphocyte responsiveness to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) or concavalin A (Con A). ADV antigen-responsive lymphocytes began to appear in the peripheral blood between 7 and 14 days after inoculation and could still be demonstrated in blood and spleen of infected pigs at 174 days after infection. In vaccinated pigs, sensitized peripheral blood lymphocytes could be detected up to at least 35 days after revaccination. Pre-incubation of ADV antigen with specific antibody markedly reduced lymphocyte stimulation. Non-immunized pigs showed no lymphocyte response to ADV antigen. Infected pigs exhibited no lymphocyte reactivity against antigens of non-infected cells.     

Cell mediated responses in a porcine enterovirus infection in piglets.

Sixteen pathogen-free piglets were infected orally with procine enterovirus strain T80 and the cell mediated response to the virus was measured at intervals after infection. Five uninfected piglets were used as controls. Indirect macrophage migration inhibition tests were performed with lymphocytes from blood, ileal lamina propria and mesenteric lymph node. Blood lymphocyte culture supernatants showed no consistent T80 specific on macrophage migration, suggesting the absence of a systemic cell-mediated response. Ileal lamina propria lymphocyte culture supernatants showed irregular migration stimulation. The mesenteric lymph node lymphocyte culture supernatants produced migration inhibition at seven days postinfection, followed by stimulation of migration from ten to 22 days postinfection. Subsequently, migration was again inhibited from 24 to 31 days postinfection. Mesenteric lymph node lymphocyte culture supernatants contained only trace amounts of interferon activity at 28 and 31 days postinfection. It was concluded that the cell mediated responses in this infection were weak and localized and not associated with significant antiviral activity.     

Cell-mediated immune response in swine infected with Mycobacterium avium subsp. avium

The zoonotic characteristic of Mycobacterium avium subsp. avium (MAA) represents a veterinary and economic problem in infected pigs. In this study, we analysed cell-mediated immunity six months after experimental infection by measuring interferon-γ (IFN-γ) production and by performing lymphocyte transformation tests after in vitro re-stimulation with the MAA-derived antigen. At the same time, IFN-γ-producing cells were characterised by flow cytometry. In MAA-infected animals, the production of IFN-γ increased in response to the MAA antigen in the blood, spleen and mesenteric lymph nodes. Similarly, a positive antigen-driven response was detected by the proliferation assay. In contrast, IFN-γ production and proliferation was undetectable after stimulation with the MAA antigen in uninfected control animals. These results indicate that both methods can be used for the identification of individual MAA-infected pigs. Using flow cytometry, we found that double-positive CD4(+)CD8(+) lymphocytes were the major T lymphocyte subset producing IFN-γ after in vitro re-stimulation.     

Detection of antibodies against Actinobacillus pleuropneumoniae serotype 5 using an inhibition enzyme immunoassay.

An inhibition enzyme immunoassay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 5 (App-5) in pig sera, based on the inhibition of the binding of an App-5 specific monoclonal antibody was established. The monoclonal antibody (MAb 210-F11) was found to be directed against an epitope on the O-chain of App-5 LPS. In the inhibition EIA, highly purified App-5 LPS was used to coat microtitre plates. Serial dilutions of pig sera were added to the plates prior to the addition of the MAb 210-F11. The degree of binding of App-5 antibodies from pig sera was determined as the percentage inhibition of the MAb 210-F11. Pig serum from specific pathogen free (SPF) herds, from experimentally infected animals, and from acutely and chronically infected herds were tested. A serum dilution of 1/30 was found to be optimal, when using 50% inhibition as the discriminating inhibition percentage. No cross-reactivity was observed with serum from pigs infected with other App serotypes or bacteria isolated from the respiratory tract, such as A. suis and H. parasuis. The inhibition EIA will be used for surveillance of App-5 antibodies in SPF and conventional herds.     

Brucella abortus antigen-induced lymphocyte reactivity in guinea pigs

A whole blood lymphyocyte transformation (WBLT) assay was used to detect anti-brucella lymphocyte reactivity in guinea pigs. Brucella antigens stimulated an antigen-specific lymphoproliferative response in WBLT assays from $\text{Brucella abortus}$ infected guinea pigs. The response was best detected from 6 to 16 weeks after challenge inoculation with viable $\text{B. abortus}$ 2308. Lymphocytes were not stimulated by unrelated bacterial antigens and control animals did not respond to the Brucella antigens. The responding cell population was characterized as mostly T lymphocytes. The WBLT assay was found to be specific for the detection of anti-brucella lymphocyte reactivity. However, a negative response was not definitive, which indicated a need for repeated testing to establish that a guinea pig did not have anti-brucella lymphocyte sensitization.     

Serological and immunological studies of pleuropneumonia of swine caused by Haemophilus parahaemolyticus

The development of circulating antibodies for H. parahaemolyticus was studied in experimentally infected SPF pigs and in-contact SPF pigs. Blood serum titers were determined by a modified complement fixation test with normal SPF swine serum as a source of supplementary complement factor, and by an indirect haemagglutination test.CF and IHA titers became positive within the first 2 weeks following exposure to H. parahaemolyticus, and reached peak values after 2 to 7 weeks (Figs. 1 to 3). The exposed pigs proved immune, in that they showed no clinical symptoms on challenge after resp. 6, 9 and 11 weeks.While distinct titers were thus obtained with both tests in SPF swine experimentally exposed to H. parahaemolyticus, the CF test proved more specific than the IHA test when the 2 tests were compared in a field outbreak of polyserositis (Glässers disease) caused by H. parasuis. The CF test would therefore seem to be preferable to the IHA test in field diagnostic work (Table 1).A noticeable finding was that challenge did not elicit an anamnestic antibody response in any of the immune pigs (Figs. 1 to 3). This fact together with negative bacteriological findings in the animals in question would seem to suggest that the challenge dose was unable to establish a permanent infection in the respiratory tract of the immune pigs.     

Studies on the role of humoral and cell-mediated immunity in pigs after vaccination with Aujeszky's disease virus

Humoral and cellular immunity in pigs vaccinated twice with Aujeszky's disease virus (ADV) was studied by seroneutralizing test and direct leucocyte migration inhibition technique. Significant migration inhibition of leucocytes (LMI) was found on the fifth day, whereas specific antibodies began to appear at that time only in very low titers. Anamnestic reaction due to the second injection of ADV did not bring about a significant increase of migration inhibition of leucocytes, instead the level of antibodies elevated markedly.     

Development of a whole blood lymphocyte stimulation assay for detecting hypersensitivity to PPD in cattle experimentally infected with Mycobacterium bovis

A whole blood lymphocyte stimulation assay utilizing the uptake of tritiated thymidine was developed for the detection of Mycobacterium bovis sensitivity in cattle. Results on eight M. bovis infected animals (six to ten weeks after infection) and eight control animals show that satisfactory lymphocyte stimulation can be obtained using heparinized whole blood diluted 10-fold in tissue culture medium and cultured with purified protein derivative (PPD) for three to seven days. Infected animals exhibited significantly greater stimulation when cultured with PPD than did control animals.     

Lymphocyte migration studies

For maintenance of immunity and tolerance, the organs and tissues of the organism are connected by migrating lymphoid cells. Understanding lymphocyte migration is essential for many disorders and diseases-- especially in the mucosa-lined organs. Detailed analyses of migrating lymphocytes have been performed in many species, especially in laboratory animals. However, important experiments in lymphocyte migration have been carried out in large animals, for example sheep, cattle and pigs. These species allow experimental procedures like in situ-organ labelling, lymphocyte retransfusion studies or lymph vessel cannulations. Such studies have made an important contribution to the understanding of the overall principles of lymphocyte migration especially in the mucosal immune system. Major results on the specific migration of na?ve and memory T cells through lymphoid organs, the re-distribution of gamma/delta T cells in the intestinal immune system and the emigration of newly produced B cells from the ileal Peyer's patches have been obtained in large animals. Since there are growing numbers of markers for large animals, and molecular biology methods are available in these species, experiments in large animals will be an essential tool for the understanding of lymphocyte migration especially in mucosal organs.     

In vitro blastogenic responses of peripheral lymphocytes from marmosets to phytohemagglutinin and to autologous lymphocytes transformed by Epstein-Barr virus

White-lipped marmosets were evaluated for their cell mediated immune (CMI) response to EBV to determine the feasibility of CMI studies in marmoset models for EBV oncogenesis. The mitogen, cell concentrations, the length of incubation period and serum requirements were defined for in vitro lymphocyte stimulation tests. The level of response of each animal was dependent on the concentration of phytohemagglutinin-P (PHA-P) and was independent of cell densities employed. The rate of tritiated-thymidine incorporation by mononuclear cells due to PHA-P increased exponentially between 2–4 days. This test was reproducible for a given batch of PHA-P when the cells were cultured in the presence of 10% heat inactivated fetal bovine serum. The five white-lipped marmosets were seronegative for EBV antigens and did not show lymphocyte stimulation with EBV particles and EBV soluble antigen, but two of these animals exhibited significant stimulation with autologous lymphocytes transformed in vitro by B95-8 virus. Despite the limited amount of blood (3–4 ml) that could be obtained from each animal in a single bleeding, it was possible to perform multiple lymphocyte stimulation assays with the protocol used.