In vivo and in vitro responses of cats sensitized with viable Mycobacterium bovis (BCG).

Cats were injected subcutaneously with viable Mycobacterium bovis (BCG), and immune responses were evaluated at various times after injection. The BCG injection produced fever, leukocytosis, neutrophilia, and lymphadenopathy of regional lymph nodes. Intradermal tuberculin injection produced responses consistent with delayed type hypersensitivity reaction in the treated cats at postinoculation day 21. Skin responses to tuberculin were not significant at postinoculation day 49. The cellular infiltrate at the tuberculin injection site at 48 hours after injection was an admixture of polymorphonuclear cells and mononuclear cells. The BCG produces strong intradermal skin responses in the cat, but the response was not long-lived as in cattle and guinea pigs. The BCG injection did not produce significant changes in the absolute total lymphocyte and absolute T-lymphocyte numbers in peripheral blood. The percentage of T-lymphocytes was significantly higher in the BCG-treated group. Differences were not observed in lymphocyte blastogenesis with tuberculin and non-specific mitogens between BCG-treated and control cats.     

Cell-mediated immune response in cattle to bovine respiratory syncytial virus

Cattle inoculated with bovine respiratory syncytial virus (BRSV) were evaluated for the development of a cell-mediated immune response. Results of the leukocyte migration-inhibition test under agarose and the delayed hypersensitivity test indicated that a cell-mediated immune response was elicited after intranasal inoculation of calves with BRSV. Migration inhibition in the leukocyte migration-inhibition test was detected by postinoculation day (PID) 5 and reached maximum inhibition on PID 21. Inhibition of leukocyte migration was still evident by PID 42 when values were still appreciably greater than preinoculation values. All of the calves inoculated with BRSV developed a delayed hypersensitivity skin response when challenge exposed intradermally with BRSV antigen.     

Mycobacteria in pond waters as a source of non-specific reactions to bovine tuberculin in New Zealand

When 71 samples were collected from ponds throughout New Zealand, 35 (49.3%) were found to contain mycobacteria. The majority of these strains (62.9%) belonged to a homogeneous group (tentative designation H-Group, which differed from any known mycobacterial species. Mycobacteria of this H-group had also been found in sphagnum vegetation growing in the immediate vicinity of many of the ponds. H-Group mycobacteria induce sensitization in guinea pigs against bovine tuberculin. The PPD sensitin prepared from these mycobacteria gave rise to larger reactions in guinea pigs than did bovine tuberculin when used in the same concentrations (500 and 50 TU). The possible sensitization of cattle to bovine tuberculin via drinking water containing H-Group mycobacteria, is discussed. The larger size of the delayed hypersensitivity reactions in guinea pigs using the same concentrations of bovine and homologous tuberculin, suggests that comparative intradermal testing might enable this non-specific reaction to be distinguished from the specific reaction developed during bovine tuberculosis infection in cattle.     

Effects of dialyzable lymph node extracts on lymphoblast proliferative capacity of blood mononuclear cells in cattle with chronic paratuberculosis.

Dialyzable lymph node extracts (DLE) containing transfer factor prepared from calves sensitized to Mycobacterium paratuberculosis and keyhole-limpet hemocyanin (KLH) were administered to 4 adult cows with chronic paratuberculosis. Cutaneous delayed hypersensitivity, lymphocyte blastogenesis, monocyte migration-inhibition, and lymphoblast proliferative capacity as a reflection of interleukin-2 (IL-2) activity were measured in response to M bovis purified protein derivative, johnin, and KLH before and after treatment with DLE. Change in cutaneous delayed hypersensitivity was not evident after DLE treatment. Alterations in histologic features of pre- and posttreatment sections of ileum and mesenteric lymph nodes were not detected. Lymph node extract treatment significantly (P less than 0.05) increased IL-2 activity and migration-inhibition in response to johnin and KLH in vitro. Treatment had no effect on lymphocyte blastogenesis. The data indicate that cattle with chronic paratuberculosis may benefit from DLE treatment, by virtue of increased IL-2 activity, and that effects of DLE are at least partially mediated by an increase in IL-2 activity.     

Mycobacteria in pond waters as a source of non-specific reactions to bovine tuberculin in New Zealand

When 71 samples were collected from ponds throughout New Zealand, 35 (49.3%) were found to contain mycobacteria. The majority of these strains (62.9%) belonged to a homogeneous group (tentative designation “H-Group”), which differed from any known mycobacterial species. Mycobacteria of this “H-Group” had also been found in sphagnum vegetation growing in the immediate vicinity of many of the ponds. “H-Group” mycobacteria induce sensitization in guinea pigs against bovine tuberculin. The PPD sensitin prepared from these mycobacteria gave rise to larger reactions in guinea pigs than did bovine tuberculin when used in the same concentrations (500 and 50 TU). The possible sensitization of cattle to bovine tuberculin via drinking water containing “H-Group” mycobacteria, is discussed. The larger size of the delayed hypersensitivity reactions in guinea pigs using the same concentrations of bovine and homologous tuberculin, suggests that comparative intradermal testing might enable this non-specific reaction to be distinguished from the specific reaction developed during bovine tuberculosis infection in cattle.     

Onset,kinetics and duration of in vivo and in vitro delayed hypersensitivity responses of neonatal calves sensitized with mycobacterial components

Newborn calves developed delayed skin-test responses 10 days after a single intradermal inoculation at birth with 150 μg of purified derivative of Mycobacterium bovis associated with 150 μg of mycobacterial immunopotentiating glycolipid P3 and oil droplets. Development of tuberculin skin-test reactivity was detected simultaneously with increased lymphocyte transformation responses. Longitudinal studies revealed that two of three calves tested at 17 months of age were still skin-test positive. The kinetics of cellular hypersensitivity responses in neonatal calves sensitized with mycobacterial components are compared to the results of acquired cellular resistance and delayed hypersensitivity studies in phylogenetically disparate species. We concluded that bovine neonates have the capacity to rapidly develop cellular immune responses following stimulation with mycobacterial antigens.     

Evaluation of bovine cutaneous delayed-type hypersensitivity (DTH) to various test antigens and a mitogen using several adjuvants

The Bacillus Calmette Guerin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of cell-mediated immune response (CMIR), specifically delayed-type hypersensitivity (DTH); however, its use in livestock may confound diagnosis of Mycobacterium tuberculosis. Therefore, various alternative antigen/adjuvant combinations were evaluated as inducers of DTH that were compared to the BCG/PPD test system with the purpose of finding a skin DTH protocol that does not cross-react with the tuberculin test and allows identification of high and low CMIR responder phenotypes. Specifically, 30 non-lactating cows (five/treatment) were sensitized on day 0 with mycobacteria [BCG, M. tuberculosis or Mycobacterium phlei cell wall extract (MCWE)], and ovalbumin (OVA) emulsified in Freund's complete adjuvant (FCA), non-ulcerative Freund's adjuvant (NUFA), complete NUFA or MCWE. On day 21, cows were injected intradermally with various test antigens including PPD tuberculin, phlein, and OVA. Phosphate buffered saline was included as the negative control and the T-cell mitogen phytohemagglutinin (PHA) was also administered. Double skin-fold thickness was evaluated before and at 6, 24, and 48 h post-injection. Skin biopsies were taken at 24 and 48 h to assess oedema, necrosis, and inflammatory cell infiltration. BCG/PPD and M. phlei/phlein treatments when given with a Freund's adjuvant induced equivalent DTH with peak reactions at 24-48 h after antigen injection. Cows receiving NUFA had fewer injection site granulomas than FCA or CNUFA treatments. The change in skin thickness response to PHA peaked at 6 h. Only cows receiving mycobacteria in NUFA had skin response to OVA, which peaked 6-24 h post-injection. Only sites tested with PPD or phlein had significantly higher lymphocyte infiltration than control, whereas neutrophils were significantly higher at PHA test sites and eosinophils predominated at the PHA test sites. Macrophages were significantly more numerous at the PPD and/or phlein test sites in treatment groups that received killed mycobacteria in a Freund's adjuvant and/or with BCG, and at the PHA test sites in all treatment groups. It was concluded that the M. phlei/phlein system did induce DTH and was similar to the DTH induced by the BCG/PPD system when MCWE was administered with a Freund's adjuvant. Therefore, this protocol is suitable for detecting high/low CMIR responders in research herds. However, cross-reaction to PPD was evident following induction of DTH using M. phlei. Hence, this protocol does not alleviate the problem of artificial induction of DTH cross-reactivity and would not be suitable for commercial herds where tuberculin testing is required.     

Immunotoxicity of ochratoxin A to growing gilts.

Ochratoxin A (OA) was incorporated in the diets of growing gilts (mean body weight, 20.1 kg) at a concentration of 2.5 mg of OA/kg of feed and was fed continuously for 35 days. Humoral and cell-mediated immunologic measurements were evaluated to determine the effects of OA on immune function in swine. Cutaneous basophil hypersensitivity to phytohemagglutinin (PHA), delayed hypersensitivity to tuberculin, PHA-induced lymphocyte blastogenesis, interleukin-2 production, total and isotype immunoglobulin concentrations, antibody response to chicken RBC, and macrophage activation were used to evaluate immune function. Gilts treated with OA had reduced cutaneous basophil hypersensitivity response to PHA, reduced delayed hypersensitivity to tuberculin, decreased stimulation index for lymphoblastogenesis, decreased interleukin-2 production when lymphocytes were stimulated with concanavalin A, and decreased number and phagocytic activity of macrophages. Differences were not observed for total and isotype immunoglobulin concentrations, or humoral hemagglutination (chicken RBC) titer. These data indicate that OA may suppress cell-mediated immune response in growing swine.     

Tuberculin reactions in bulls and boars sensitized with atypical Mycobacteria from sawdust.

Due to a planned export from a combined bull and boar station, more than 70 boars and 100 bulls were examined by tuberculin tests. Distinct reactions to avian tuberculin appeared in about half of the animals. Many of them also reacted to bovine tuberculin. For diagnostic purposes, many of the reactors were slaughtered, and samples from these and from the environment were examined bacteriologically. Strains of Mycobacterium avium were isolated from only 2 out of 14 reacting boars and from none of the 23 reacting bulls. No isolation of Mycobacterium bovis was made. However, atypical mycobacterial strains, classified as Runyon Group III and IV, were isolated from 3 boars, 2 bulls, 1 pigeon and from many samples of sawdust. The isolation of identical fast-growing mycobacterial strains from the sawdust used in the pens for the reacting boars and bulls, was especially remarkable. The strains differed enzymatically and biochemically from those isolated from other sources. This indicated a possible sensitization of the animals with similar mycobacterial strains. Possible cross-reactions to avian and bovine tuberculin were investigated in tuberculin assays with guniea pigs and pigs sensitized to one of the mycobacterial strains isolated. Distinct reactions to both avian and bovine tuberculin appeared in all the animals. From these results it was concluded that the tuberculin reactions in the boars and the bulls were not due to any tuberculous infection in the herd, but to a sensitization of the animals with atypical mycobacteria.     

Experimental infection with Mycobacterium avium, serotype 2, in pigs. 1. Intravenous inoculations

A porcine strain of Mycobacterium avium, Serotype 2, was used for intravenous inoculation of pigs in doses 5, 1, 10⁻¹, 10⁻² and 10⁻³ mg (1 mg = 78 × 10⁶ viable units), 2 pigs per dose.Dose 5 mg proved fatal for both of the inoculated pigs, which were killed in extremis 64 and 69 days, respectively, after inoculation. Dose 1 mg caused clinical disease in 1 of 2 pigs, but was not lethal. Post mortem, the clinically affected pigs showed a generalized granulomatous tuberculosis. The other pig given 1 mg and the pigs given smaller doses, showed no clinical signs, and lesions and presence of acid-fasts were mostly limited to the lymph nodes of the lung, liver and digestive tract.All the pigs showed delayed hypersensitivity to avian PPD tuberculin (1000 t.u.) and some of them cross-reacted with human PPD tuberculin (1000 t.u.). The clinically affected pigs gave a very weak response to tuberculin, the others a strong response.The smallest dose capable of establishing an infection and producing tuberculous lesions was not determined, but seems to be less than 10⁻³ mg (78000 viable organisms).     

Chronic superficial keratitis in dogs: detection of cellular hypersensitivity.

Dogs affected with chronic superficial keratitis (CSK) and clinically normal dogs were tested for cellular hypersensitivity, using the leukocyte migration-inhibition (LMI) technique to 3 ocular antigens (Staphylococcus aureus and corneal and iridal proteins). Affected dogs had statistically significant increases in hypersensitivity cellular responses against corneal and iridal antigens. Affected dogs did not differ from clinically normal dogs in their cell response to S aureus.     

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.     

The prevalence of immediate and delayed type hypersensitivity reactions to Microsporum canis antigens in cats

Spontaneous recovery from Microsporum canis infections in cats is thought to be dependent on the development of a competent immune response. The purpose of this study was to determine the prevalence of positive delayed type hypersensitivity reactions in cats with and without dermatophytosis. Four groups of cats were intradermally skin tested with M canis extract and test sites were evaluated both subjectively and objectively at 0, 24 and 48 h after injection. Delayed intradermal testing (IDT) reactions were absent in cats not exposed to dermatophytosis (n=20); infected-recovered cats (n=38 culture negative lesion negative and n=43 lesion negative but culture positive) had significantly larger IDT reactions than unexposed cats and cats that were still actively infected (n=18). Based on the results of this study, IDT with M canis extract can be used to assess the cellular immune response of cats with dermatophytosis.     

Specific suppression of the bursa-dependent immune system of chicks with infectious bursal disease virus.

Chicks which had been inoculated with infectious bursal disease virus (IBDV) at 1 day of age had a severe depression of bursa-dependent humoral immune functions by day 42. Antibody responses against rabbit red blood cells or to immunization with bovine serum albumin were significantly suppressed. In contrast, chicks inoculated with IBDV at 21 days of age produced near normal antibody responses as compared with the responses in noninfected control chicks. The IBDV had no significant effect on the thymus-dependent cellular responses as measured by skin graft rejection or delayed type hypersensitivity reactions to tuberculin.     

Epidemiologic investigation of Mycobacterium bovis in a population of cats

OBJECTIVE: To determine whether cats exposed at a residence were infected with Mycobacterium bovis, whether the tuberculin skin test can identify cats infected with M bovis, and whether an ELISA could identify tuberculosis-infected cats. ANIMALS: 20 domestic cats exposed to a cat with laboratory-confirmed disseminated M bovis infection. PROCEDURE: Cats were administered a tuberculin skin test and monitored for 72 hours. Blood and fecal samples were collected. Cats were then euthanatized, and postmortem examinations were performed. Tissues were examined grossly and histologically for signs of mycobacteriosis. Pooled tissue samples and fecal samples were submitted for mycobacterial culture. Blood samples were examined for evidence of tuberculosis by use of a comparative ELISA. RESULTS: 4 cats had positive responses for the ELISA, and 2 cats had suspicious responses. All tuberculin skin tests yielded negative results. No gross or histologic lesions of tuberculosis were detected in any tissues, and mycobacteria were not isolated from tissues or feces obtained from the 20 cats. CONCLUSIONS AND CLINICAL RELEVANCE: All cats that had positive or suspicious responses for the ELISA were offspring of the cat with tuberculosis. Evidence of tuberculosis was not seen in other cats at the residence, the owner, or the attending veterinarian. The most likely source of tuberculosis for the infected cat was through the consumption of M bovis-infected wildlife carcasses or offal. Because M bovis is endemic in wildlife in northeastern Michigan, there is a risk of exposure to tuberculosis in companion animals, their owners, and attending veterinarians.     

Immune responsiveness and lymphoreticular morphology in cattle fed hypo- and hyperalimentative diets

Immune responses and lymphoreticular morphology were studied in 3 groups of yearling Hereford steers fed hypoalimentative, maintenance and hyperalimentative diets for 142 days. Significant decreases in plasma protein levels and circulating lymphocyte levels occurred in low energy intake steers. Percent circulating lymphocytes bearing surface immunoglobulins and serum levels of IgG and IgM did not vary significantly within or between groups. Antibody responses to Brucella abortus bacterin inoculated on day 63 were similar in all 3 groups. Antibody responses to chicken erythrocytes inoculated on day 88 were significantly lower in hypoalimentated steers. Differences between groups in lymphocyte blastogenic responses to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were not significant. Hyperalimentated steers had significantly depressed PWM responses compared to a baseline value established for that group. In addition, hypoalimentated steers tended to have elevated responses to PHA, although differences were not significant. There were no significant differences between groups in dermal hypersensitivity responses to tuberculin following sensitization with viable Bacillus Calmette-Guerin (BCG). The thymuses of hypoalimentated steers were markedly atrophied but lymph nodes and splenic white pulp were normal. Thymus, lymph nodes and spleen were normal in hyperalimentated steers.     

Lack of immunostimulatory effect of N-acetyl muramyl-l-alanyl-d-isoglutamine on selected chicken immune functions

Synthetic N-acetyl muramyl-l-alanyl-d-isoglutamine, syn. muramyl-dipeptide (MDP) was found to be immunostimulatory in several experimental animal species. In order to determine the influence of MDP on the chicken immune response, different doses (0.05–0.2 mg) of this compound were administered to 6-week old chickens, and cellular as well as humoral immune functions were tested. Neither the immune response against sheep red blood cells or Newcastle disease virus (strain Hitchner B 1), nor the ability to reject skin grafts, or to react in the delayed hypersensitivity (tuberculin) test, were affected significantly under the experimental conditions employed. This study reveals little evidence for parallels between the ability of the chicken immune system and the immune system of other animal species examined so far, to develop enhanced immune reactions under the influence of MDP.     

Evaluation of comparative cervical tuberculin skin testing in cervids naturally exposed to mycobacteria

A comparative cervical tuberculin skin test was evaluated in cervids with naturally acquired mycobacterial infections. The test exhibited a high degree of sensitivity (84%) and specificity (80%) in detecting infected animals but a low level of accuracy (57%) in differentiating Mycobacterium bovis infections from infections caused by M avium and other Runyon groups III and IV mycobacteria. This phenomenon was attributed to a low recovery rate of more than 1 species of mycobacteria from individuals with mixed infections.     

Immunologic skin tests in piglets

Immunological skin tests were carried out in 213 weanling piglets at the weight of 5 to 30 kg. Immediate hypersensitivity was evaluated by help of diagnostic allergens (mould, yeast and bacterial ones), delayed hypersensitivity by help of staphylococcus lysate, cellular immunity (tests de novo) by help of phytohemagglutinin and as common recall antigens were used tuberculin, toxoplasmin, candidic and tetanic antigen. The above substances were applied intradermally to the back of the piglets. For an evaluation of non-specific inflammatory response, sodium lauryl sulphate applied epicutaneously was used. The average reaction to diagnostic allergens (50 PNU in 0.05 ml) evaluated after 20 minutes was characterized as light dermal reactions (the papule size of 3-5 mm); in 22% of piglets moderate dermal reactions (the papule larger than 6 mm) to the diagnostic bacterial allergen (Staphylococcus aureus, Staphylococcus epidermidis) were determined. After an intradermal implantation of histamine (50 micrograms in 0.05 ml) the average size of the papule was 13 mm in 20 minutes. After the application of 0.1 ml of staphylococcus lysate (STAVA), the induration exceeding 10 mm was observed in 13% of piglets in 24 hours. After the application of phytohemagglutinin (100 micrograms in 0.1 ml), the induration exceeding 5 mm in 24 hours was determined in 93% of piglets and in 48 hours in 59% of piglets. Common recall antigens were applied at the volume of 0.1 ml. Forty-eight hours after the application no palpable induration was determined: in 91% of piglets after the application of tuberculin (2 TU PPD in 0.1 ml); in 75% of piglets after toxoplasmin (according to PNY 30-33-74); in 98% of piglets after candidic antigen (100 PNU in 0.1 ml) and in 86% of piglets after tetanic antigen implantations (0.03 Lf in 0.1 ml). The epicutaneously applied sodium lauryl sulphate (at 2.5% and 5% concentrations) caused no inflammatory dermal reactions (erythema or induration) after 24 hours. In piglets it is best to apply phytohemagglutinin for evaluating cellular immunity, staphylococcus lysate for evaluating delayed hypersensitivity to staphylococci and histamine for obtaining the information on a disposition of piglets to allergic diseases.     

Miliary dermatitis in the cat

Miliary dermatitis is a cutaneous response to disease states seen in the cat. It is not in itself a diagnosis. Conditions which may lead to miliary dermatitis lesions can be divided into hypersensitivity disorders, parasitic causes, infections and miscellaneous causes. A thorough investigation frequently leads to a diagnosis which enables specific therapy. Symptomatic, empirical therapy is less satisfactory and may in the long term cause side effects, The single most important cause of miliary dermatitis in the UK and elsewhere in the world is flea-bite hypersensitivity. Other important and increasingly recognised causes include atopy and food hypersensitivity. With determination and a thorough approach to diagnosis the number of idiopathic cases of miliary dermatitis are small.