In vivo function tests of the effect of tilorone and niridazole on cell-mediated immunity in chickens.

The need for effective, safe, specific cellular immune suppression in avian research led to the study of effects of tilorone and niridazole on cell-mediated immunity of chickens. Two in vivo tests for cell-mediated immunity function were used--the graft-vs-host (GvH) test and the delayed hypersensitivity (DH) test. Humoral immunity was evaluated by measuring natural hemagglutination (HA) titers against rabbit red blood cells. Intraperitoneal administration of tilorone to young chickens appeared to have severe toxic side effects and was of little value as an immune suppressant. Oral administration of tilorone to 6-week-old chickens caused DH suppression, but no marked effect was seen on GvH reactions or HA titers. Toxicosis appeared less severe. Oral administration of niridazole to 6-week-old birds caused nearly complete loss of GvH and DH reactivity but caused an increase in HA titers. General toxic effects of niridazole were not apparent.     

Cellular and humoral response of in ovo-bursectomized chickens to experimental challenge with velogenic Newcastle disease virus

Humorally deficient, in ovo-bursectomized (Bx) and sham-Bx chickens were vaccinated twice, 1 month apart, with Newcastle disease virus (NDV) Roakin strain and challenged with a velogenic viscerotropic NDV strain via the oronasal route. Hemagglutination-inhibition and seroneutralization tests showed that Bx chickens had reduced antibody-mediated immunity to virus infection. In contrast, they had significantly higher cell-mediated immunity (CMI) before challenge, as estimated simultaneously by determination of blastogenic capacity of peripheral blood lymphocytes induced by phytohemagglutinin and by specific antigen stimulation. After virus challenge, there was transitory inhibition of CMI based on marked reductions in levels of stimulation indices, and this impairment in CMI was supported by persistence of virus in Bx chickens for longer periods. Bx chickens resisted challenge, even though antibody titers were well below those considered predictive of resistance to challenge, suggesting that CMI provides a degree of resistance to velogenic NDV.     

Interdigital skin test for evaluation of delayed hypersensitivity and cutaneous basophil hypersensitivity in young chickens

A skin test to assess T-cell mediated delayed hypersensitivity (DH) and cutaneous basophil hypersensitivity (CBH) was evaluated in the interdigital skin of young chickens. Three-day-old chickens were sensitized with Mycobacterium tuberculosis, and the DH reaction was elicited in the interdigital skin in 10-, 17-, 24-, and 31-day-old chickens by intradermal injection of tuberculin. Cutaneous basophil hypersensitivity was elicited in the interdigital skin of 10- and 14-day-old chickens by a single intradermal injection of phytohemagglutinin-P (200 micrograms). The effect of immunosuppression on the results of interdigital skin test for DH and for CBH was evaluated in chickens that were treated with dexamethasone daily for 4 days before testing. The DH reaction, as indicated by a significant (P less than 0.01) increase in the mean interdigital skin thickness, was detectable in 10-day-old chickens and was consistently evident in 17-, 24-, and 31-day-old chickens. The DH response in the interdigital skin of 24-day-old chickens was comparable with that elicited in the standard wattle test. The CBH reaction, as indicted by a significant increase (P less than 0.005) in skin thickness, was evident in the interdigital skin of 10- and 14-day-old chickens. Treatment with dexamethasone significantly decreased (P less than 0.01) the DH and CBH reactions. Results of the study indicated that the interdigital skin test may be used to evaluate normal and suppressed cell-mediated DH and CBH reactions in chickens as young as 10 and 14 days old.     

In vitro assays of cell-mediated immunity in calves with persistent Mycobacterium avium infections do not correlate with delayed skin hypersensitivity

A long-term kinetic study of the immune response of four calves experimentally infected with a M. avium strain was made using the following tests: lymphocyte stimulation (LS), leukocyte migrationinhibition (LMI), delayed skin hypersensitivity (DHS), and a radioimmunoassay (RIA) procedure for antibodies to M. avium. The cell-mediated immune (CMI) responses measured by these tests showed different courses during the infection period. The LS test showed several periods of peak values and the LMI test two peaks of responses of relatively shorter duration. At the end of the experimental period the DHS responses had decreased to insignificant levels, whereas the lymphocytes from all the calves responded in the LS test. No correlation could be detected between CMI and antibody mediated responses, and only two of the infected calves showed clear cut antibody responses measured by RIA. The results probably reflect the measurement of different aspects of the CMI response in the different test systems. The cyclical nature of the CMI measured by the LS and LMI tests may indicate the influence of regulatory mechanisms by suppressing lymphocyte sub-populations.     

Evaluation of the cell-mediated immune response in cattle induced by 2,4-dinitrochlorobenzene

A technique has been evolved and standardized for evaluating the cell-mediated immune (CMI) response in cattle induced by 2,4-dinitrochlorobenzene (DNCB). Six healthy cross-bred Jersey cows were used. The dose and the number of applications for primary sensitization and the challenge dose after the 14th day have been standardized. The gross and histological pictures of the CMI response shown by these cattle are described in detail and their usefulness in assessing the CMI response in various disease conditions including tumour is indicated.     

Correlation of in vitro and in vivo tests for cell-mediated immunity in the dog

Using tuberculin (purified protein derivative) as the test antigen, 29 dogs with different vaccination histories were tested with the lymphocyte transformation (LT) assay, the indirect agarose leukocyte migration inhibition (LMI) assay, and the skin test for delayed type hypersensitivity. All three tests were done simultaneously on each dog. The LT assay results were found to correlate well (r = 0.88) with the skin test results, whereas LMI results were found to correlate poorly (r = 0.55) with the skin test results. It was concluded that the LT assay is a more reliable measure of cell mediated immunity in the dog than is the LMI assay.     

Skin test and gamma interferon enzyme-linked immunosorbent assay results in sheep exposed to dead Mycobacterium avium subspecies paratuberculosis organisms.

Cell-mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) and the Johnin skin test, have the potential to detect animals infected with Mycobacterium avium subspecies paratuberculosis (MAP) early in the course of the disease. While these CMI tests tend to be relatively specific in noninfected flocks, in MAP-infected flocks, these tests often identify animals that cannot be confirmed infected by any other reference test, including necropsy and culture. The aim of this study was to determine if antigen exposure by inhalation or oral ingestion of killed MAP organisms would cause a detectable CMI response in sheep. Forty-eight lambs 4 months of age were randomly divided into a control group, an orally exposed group (dosed with 1 x 10(10) autoclaved MAP organisms 3 times), and an inhalation-exposed group (dosed once with 1 x 10(5) dead organisms). Lambs were skin tested and/or bled pre-exposure and 1, 2, 3, 4, and 12 months postexposure. No significant difference was seen with either the oral- or inhalation-exposed groups of lambs versus controls with either the IFN-gamma ELISA or the skin test at any time pre- or postexposure. These results suggest that infection/invasion of MAP organisms must occur in order to have a positive skin test or IFN-gamma ELISA beyond the false-positive rate. Simple exposure is not enough to elicit a detectable CMI response.     

The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis

The comparative intradermal skin test, in which a delayed type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD) from Mycobacterium bovis and M. avium is assessed and compared, may be used repeatedly on non-infected animals on farms where bovine tuberculosis (TB) has occurred. A skin test is known to affect subsequent skin tests in infected animals. The reported study was to determine whether repeated skin testing prior to infection with M. bovis might affect the development of the comparative skin test and IFNgamma response subsequent to exposure to virulent M. bovis. The comparative intradermal skin test was applied to one group of six calves five times at 8-week intervals. These and six control calves were subsequently inoculated intratracheally with a dose of M. bovis that produced mild disease. The development of the DTH reaction, IFNgamma, IL-10 and proliferative responses were compared in the two groups of animals. No differences in IFNgamma, IL-10 and proliferative responses were seen between the two groups of calves prior to challenge. After infection with M. bovis no differences in the development of the DTH and IFNgamma responses to PPD were noted as a consequence of the repeated skin testing prior to challenge. No differences between the groups were evident when ESAT-6 was used as antigen and IFNgamma was assayed, although two animals that responded to PPD did not respond with ESAT-6. However, there did appear to be subtle effects of repeated skin testing on the immune response post-challenge that did not affect the diagnostic tests. After challenge control animals showed greater proliferative responses than animals given repeated skin tests prior to challenge, indicating that the procedure did have consequences for immune responses following infection. In both groups a marked reduction in the intensity of the skin test and in the number of animals that would be recognized as reactors was evident when animals were tested 15 weeks post-infection compared to their responses 8 weeks earlier that could have consequences for diagnosis of TB. An antibody response was not evident as a result of repeat skin testing prior to infection but was seen in both groups of calves following skin testing performed 7 weeks after infection.     

Laryngotracheitis in chickens: the length of the preinfectious and infectious periods

Transmission studies to measure the length of the infectious period and the interval between virus inoculation and infectiousness were conducted using the standard National Veterinary Services Laboratory laryngotracheitis (LT) challenge virus (Log 10(6.7) EID50 per ml). Previously unexposed sentinel chickens were placed in contact with chickens inoculated intratracheally with LT virus. Transmission of virus to the sentinel birds was assessed by studying clinical signs and results of virus isolation and challenge. Chickens began to shed infective quantities of virus 2-4 days postinoculation and continued until day 6.     

Immune Response to other Agents of Calves Persistently Infected with Bovine Virus Diarrhoea Virus (BVDV)

Houe, H. and I. Heron: Immune response of calves persistently infected with Bovine Virus Diarrhoea Virus (BVDV) to other agents. Acta vet. scand. 1993, 34, 305-310.– The ability of calves persistently infected (PI) with bovine virus diarrhoea virus BVDV to respond immunologically to defined antigens other than BVDV was studied. Five clinically healthy PI calves were studied together with 5 non-PI calves serving as controls. The humoral immune response was tested by measuring the serum antitoxin titre following immunization against tetanus. The cellular immune response was tested by the ability to develop a positive reaction in a cutaneous tuberculin test performed 1 month after immunization against Johne’s disease (paratuberculosis). Finally, a skin-sensitizing agent, dinitrochlorobenzene (DNCB), was employed to study whether PI calves would react by hypersensitization following skin exposure to DNCB for 7 consecutive days followed by application of DNCB to a new skin area remote from the area that had first been exposed.The response of PI calves to the various types of antigenic stimuli applied was not significantly different from that of the control calves. Thus, PI calves developed a potent antitoxin response after tetanus immunization, they showed a positive reaction to tuberculin skin test after immunization against paratuberculosis, and were skin sentitized with DNCB.     

Delayed-type skin hypersensitivity and in vitro lymphocyte immunostimulation responses of swine following inoculation with Mycobacterium avium cell walls and a mycobacterial immunopotentiating glycolipid

Miniature swine (n = 5 per group) were inoculated intradermally with mineral oil-in-water emulsions containing either 150 μg of mycobacterial immunopotentiating glyco-lipid P3 (EP3), 150 μg of lyophilized Mycobacterium avium (serotype 8) cell walls (E-MaCW), or 150 μg P3 and 150 μg M. avium cell walls (EP3-MaCW). Swine vaccinated with E-MaCW and EP3-MaCW developed antigen-sensitive lymphocytes detectable with delayed-type hypersensitivity (DTH) skin tests and lymphocyte transformation assay. Swine injected with EP3 were not sensitized. In general EP3-MaCW evoked a more pronounced in vivo DTH tuberculin skin test and in vitro lymphocyte transformation responses than E-MaCW. Time-course studies indicated a more persistent response in swine injected with EP3-MaCW than in those given E-MaCW. Commercial type Yorkshire swine (n = 5) inoculated intradermally with EP3-MaCW developed cell-mediated immune (CMI) responses to avian tuberculin detectable in vivo with delayed-type skin hypersensitivity and in vitro with lymphocyte immunostimulation responses.     

Suppression of cell-mediated immunity by purified aflatoxin B1 in broiler chicks

The effect of purified aflatoxin B1 on cell-mediated immunity (CMI) in broiler chicks was assessed using doses of 0.3 and 1 mg/kg feed from hatching to 6 weeks of age. Total lymphocyte and T lymphocyte counts and the 2,4-dinitrofluorobenzene skin sensitivity test, graft-versus-host reaction and nitroblue tetrazolium salt reduction tests were used to evaluate CMI. Both doses of aflatoxin B1, including the apparently nontoxic dose of 0.3 mg/kg feed, caused a significant (P less than 0.05) decline in CMI. The functional activity of splenic macrophages was decreased significantly (P less than 0.05) by both doses of the toxin.     

Suppressor activity of bovine Fc gamma positive cells during a persistent infection with Mycobacterium avium

The cell mediated immune response (CMI) was measured in calves after experimental infection with Mycobacterium avium. Using the tuberculin skin test a CMI response could be measured from four to 14 weeks after infection, and with a lymphocyte stimulation (LS) test from six to 40 weeks. One year after infection no CMI response was detected by either of the tests, in spite of the fact that in such calves M avium bacteria could be found in the intestinal lymph nodes at autopsy. After removal of mononuclear cells bearing receptors for the Fc part of IgG, the peripheral blood lymphocytes obtained from a calf infected one year earlier responded to M avium pure protein derivative in the LS test in contrast to lymphocytes obtained from uninfected calves.     

Enhanced cutaneous hypersensitivity reactions are associated with ovine high and low cortisol responsiveness to acute endotoxin challenge

Inbred rodent studies have demonstrated that cutaneous hypersensitivity reactions are exacerbated in stress-susceptible, and attenuated in stress-resistant strains of mice. This physiological response was, in part, mediated by activation of the hypothalamic-pituitary-adrenal axis during the acute restraint stress. A study was conducted to examine whether or not cutaneous hypersensitivity reactions are also associated with variable cortisol responsiveness to inflammatory stress in an outbred ovine population. High (H), medium (M), and low (L) cortisol responsive sheep were identified from a population of 110 females based on their estimated breeding values for cortisol concentration measured 4 h post-systemic challenge with Escherichia coli endotoxin (400 ng kg(-1)). Cutaneous hypersensitivity reactions to phytohaemagglutinin (PHA), 1-chloro-2, 4-dinitrobenzene (DNCB), and Candida albicans cellular antigen (CAA) were measured in these variable cortisol-responding sheep, in addition to serum interleukin (IL)-6, interferon (IFN)-gamma, and ovalbumin (OVA)-specific IgG concentrations. When compared to the M cortisol responders, both H and L cortisol responders had significantly greater cutaneous swelling during the elicitation phase in response to DNCB (P < 0.05) and CAA (P < 0.05); a similar but not significant trend was observed during the PHA challenge. The primary, but not the secondary, IgG response to OVA was significantly lower in the H and L cortisol responders when compared to the M cortisol responders. Differences in serum IL-6 or IFN-gamma concentration were not observed across variable cortisol-responsive groups. Together, these results demonstrate that cutaneous hypersensitivity reactions are enhanced in outbred H and L cortisol-responding sheep, independent of systemic modulation by IL-6 and IFN-gamma.     

Histochemical identification of tissue eosinophils in the inflammatory response of the fowl (Gallus domesticus)

A staining technique was developed for the differential identification of tissue eosinophil and heterophil leucocytes in the fowl. Pieces of formalin-fixed skin, challenged with dinitrochlorobenzene (DNCB) or citraconic anhydride (CA), were incubated in a substrate suitable for peroxidase prior to embedding in either paraffin wax, glycol methacrylate or Araldite. This results in deep brown staining of the eosinophil granules while those of the heterophils remain unstained. Heterophils and eosinophils were conspicuous at 30 minutes after challenge in the early inflammatory response. By 48 hours the heterophilic response had diminished and eosinophils had almost disappeared. Only mononuclear cells were seen at 72 hours. It is suggested that the eosinophil leucocyte might act as an early modulator of inflammation in delayed-type hypersensitivity responses in the fowl.     

Genetics of immunocompetent traits in a synthetic broiler dam line

Three hundred and three chicks of both sexes, from a synthetic dam line (SDL) of broiler chickens, were studied for economic traits (body weights at 4, 5 and 6 weeks of age) and immunological traits (humoral and cell mediated immune responses, and serum lysozyme concentration). The objective was to evaluate these traits and to estimate their genetic and non-genetic parameters. The humoral immune response was assessed by estimating the antibody response to sheep red blood cells using the haemagglutination (HA) test and serum IgG concentration using single radial immunodiffusion (SRID). The cell mediated immune (CMI) response was estimated as in vivo response to a mitogen (PHA-P). Serum lysozyme was measured by lysoplate assay. Least squares means for body weight at 4, 5 and 6 weeks were 684 +/- 20, 920 +/- 19 and 1205 +/- 28 g, HA titre was 6.289 +/- 0.246, CMI was 0.438 +/- 0.015 mm, lysozyme was 1.860 +/- 0.047 microg/ml and IgG was 6.287 +/- 0.194 mg/ml. There was an effect of sire on HA titre and on body weight at 4, 5 and 6 weeks of age; males were heavier than females. Heritability estimates were high for body weights but low for immunological traits. Phenotypic correlations (rp) among body weights were high and positive but were very low between body weights and most immunological traits. Among the immunological traits all rp were very low. Genetic correlations (rg) of body weights were positive and medium to high with CMI and HA and negative with serum IgG.     

Effect of repeated intradermal injections of bovine herpesvirus type 1 antigen on seronegative cattle.

Forty-three cattle seronegative to bovine herpesvirus-1 (BHV-1) were given from one to five intradermal injections of BHV-1 inactivated antigen at four-week intervals. This delayed hypersensitivity test was assessed by the increase in skin thickness. The activity of the antigen was assessed in five animals which had a previous natural BHV-1 infection with clinical signs and seroconversion. Anti-BHV-1 antibodies were detected by seroneutralisation and an enzyme-linked immunoassay. Only one animal showed a significant but slight increase in skin thickness after the first test, but it was negative after a second test. The animals remained seronegative after the first test. Seroconversion was identified in 11 of the 43 animals (25 per cent) submitted to repeated delayed hypersensitivity tests. Five of 37 animals seroconverted after only two tests. The serological response was transient in seven of 11 seroconverted calves. Repeated hypersensitivity tests were therefore able to induce a serological response in seronegative calves but the response was weak and often transient. The test must therefore be applied cautiously to seronegative animals.     

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression.     

Evaluation of cell-mediated immunity during chronic organophosphate pesticide intoxication in mice and goats

The status of cell-mediated immunity (CMI) after pesticide exposure was assessed in mice with the help of skin sensitivity and graft versus host reaction tests. It was observed that at 24 hours post-challenge CMI values did not differ significantly from the control, indicating no effect of quinalphos treatment in mice. Goats receiving monocrotophos at a dose rate of 1.0 mg kg-1 body mass for 40 days gave a similar result when CMI was tested with the help of the chemical sensitizer dinitrofluorobenzene (DNFB). Thus the results clearly indicate that the tested organophosphates do not interfere with cellular immunity in the intoxicated animals.