K.E. Müller

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.

Impaired specific immunoreactivity in cows with hepatic lipidosis.

In this study, hepatic lipidosis in cows was experimentally induced by offering an energy surplus during the dry period. Liver triacylglycerol (TAG) was 16% in the experimental group. In the control group fed the same diet in restricted quantities, liver TAG was about 7%. The animals of both groups were vaccinated with tetanus vaccine at Day 3 after parturition. It was demonstrated that the cows with high liver TAG percentages had lower humoral and cellular (P < 0.05) immunological responses compared with the animals with low liver TAG levels at Day 14 after vaccination. The results obtained in the high TAG group support the notion that the frequent occurrence of aspecific infections in cows with hepatic lipidosis may be due to impaired immunoreactivity.

Mycobacterium paratuberculosis heat shock protein 70 as a tool in control of paratuberculosis

Paratuberculosis in cattle is a chronic intestinal disease in which a distinctive cellular reactivity of a Th1-type preceeds the phase in which antibody titers are easily detectable and the animal becomes clinically ill. During infection with Mycobacterium avium ssp. paratuberculosis (M.a.p), a decrease in CD4 T-helper cells has been observed in the clinical phase. Our ultimate aim is to elicit a cytotoxic reaction against infected macrophages, using recombinant Hsp70 (rHsp70) of M.a.p. as a tool to shuttle antigen into the MHC class I antigen presentation pathway. To investigate the mechanism of rHsp70 as a carrier for antigen into the cell, we studied the interaction between APC and Fitc-labelled rHsp70, using FACS analysis and confocal microscopy. Interaction of rHsp70 with the cell surface of bovine APC, presumably via a receptor, was shown on monocytes, monocyte derived macrophages and dendritic cell (DC). The interaction is detectable on the complete population of freshly derived monocytes, although peak intensity of fluorescence is lower on these cells than on macrophages and DCs. DCs show interaction on a high percentage of the cells, with high intensity, while in the case of macrophages only a subpopulation interacts with rHsp70. Efficient uptake of rHsp70 as compared to OVA is shown. Preincubation of DC with unlabelled rHsp70 leads to a decreased interaction with rHsp70-FITC. DC interacting with rHsp70 in addition showed high expression of MHC I, MHC II, Myd-1 (CD172a) and CD40. Further research will focus on loading of the rHsp70 with M.a.p. antigen for presentation in MHC class I.

Bovine leukocyte adhesion deficiency--clinical course and laboratory findings in eight affected animals.

The clinical course of Bovine Leukocyte Adhesion Deficiency (BLAD) in eight Holstein Friesian cattle is described. Affected animals were presented with a history of poor thriving and recurrent bacterial infections. Five of these animals had to be killed because of severe respiratory disease shortly after admittance. Three affected animals survived calfhood only as a result of frequent antibacterial treatments. At one year of age, failure to thrive and stunted growth were still evident, but infections requiring antibiotic treatments occurred only sporadically. Clinical manifestations of BLAD were found in the digestive system (gingivitis, periodontitis, alveolar periostitis, diarrhoea), the respiratory system and the skin (impaired wound healing, chronic dermatitis). A leukocytosis based on a mature neutrophilia, which persisted during infection-free periods, was observed in all animals. Granulocytes were substantially deficient of beta 2-integrin expression on their membranes. Anaemia, which was noted in four animals, may be related to the Anaemia of Inflammatory Disease Complex (AID). The serum total protein content increased with time and was associated with elevated gamma-globulin levels. We suggest that, at a certain age, animals affected with BLAD are able to cope with environmental agents due to compensatory mechanisms of the immune system.

Neutrophil migration in the lung, general and bovine-specific aspects.

Lung inflammation is often associated with sustained neutrophil migration into the lung tissue, causing undesired side effects, i.e. substantial damage of lung tissue and fibrin deposition, which hamper complete recovery. The need for additional anti-inflammatory treatment strategies focused attention on the function of cellular adhesion molecules (CAMs) on the leukocyte membrane, which guide migration of leukocytes across the endothelium to the site of inflammation. Recent data indicate that neutrophil migration in the lung is mediated by unique pathways, involving different CAM as compared to other organs. These pulmonary characteristics of neutrophil migration enable specific targeting of CAM for anti-inflammatory treatment of pneumonia. In addition, the recent interest in intracellular signaling revealed that most CAM not only function in adhesion, but also play an important role in initiation of intracellular signal transduction and vice versa may be modulated by intracellular signaling molecules (ISMs). In this review, the mechanisms of neutrophil migration in the lung and the role of ISM with respect to CAM function are described in the context of potential anti-inflammatory intervention strategies.

Post‐mortem findings in calves suffering from bovine leukocyte adhesion deficiency [BLAD)

Five female Holstein-Friesian calves were clinically suspected of suffering from Bovine Leukocyte Adhesion Deficiency (BLAD), because of multiple recurrent infections and persistent leucocytosis. The diagnosis was established by a Polymerase Chain Reaction (PCR) technique, by which a point mutation in the DNA encoding for the CD18 allele was detected. The animals either died spontaneously or were killed because of incurable life threatening infections. Gross post-mortem examination revealed severe and extensive necrotizing processes mainly located in the respiratory and digestive tract. Microscopic examination of these necrotizing lesions demonstrated a lack of extravascularly located polymorphonuclear granulocytes (PMN) although vascular leucocytosis was obvious. However, extensive catarrhal bronchopneumonia was noticed with marked infiltration of the alveoli and bronchioli by PMN. These findings are discussed in relation to the pathogenesis of BLAD.

Identification of monoclonal antibodies with specificity to α- or β-chains of β2-integrins using peripheral blood leucocytes of normal and Bovine Leucocyte Adhesion Deficient (BLAD) cattle

Abstract The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant Leucocyte Antigens with lymphocytes and monocytes of normal and β 2 -integrin deficient (Bovine Leucocyte Adhesion Deficiency: BLAD) animals was determined by flow cytometry to investigate potential specificities to α- or β-chains of β 2 -integrins. From the 13 monoclonal antibodies that were entered as having specificity for CD11/CD18 antigens, ten were confirmed correct, but three had reactivity with cells from BLAD animals. We conclude that our approach provides an easy way to reliably identify the majority of β 2 -integrin specific monoclonal antibodies.

Identification and Phenotyping of Leukocytes in Bovine Bronchoalveolar Lavage Fluid

ABSTRACT A method is proposed to identify leukocyte subpopulations in bovine bronchoalveolar lavage fluid by dual-laser flow cytometry. The technique uses several parameters, i.e., exclusion of highly autofluorescent alveolar macrophages and inclusion of leukocytes on the basis of labeling by specific antibodies and light scatter characteristics.

Reduced lymphoid response to skin allotransplants in cows with hepatic lipidosis

The immune responsiveness of cows with hepatic lipidosis (fatty liver) in comparison to control cows with a normal liver fat content was tested by applying skin allotransplants to the skin of the back of cows on day 3 after parturition. Immunoreactivity was determined by semiquantitative counting of the number of infiltrating lymphocytes in the recipient skin adjacent to the allotransplants during a period of 21 days. There were more invading lymphocytes in samples from control cows than there were in samples from cows with hepatic lipidosis. It was concluded that cows with hepatic lipidosis have a reduced lymphoid response to skin allotransplants.

ALLOGRAFT REJECTION IN CATTLE WITH BOVINE LEUKOCYTE ADHESION DEFICIENCY

In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion deficiency (BLAD) was studied by means of skin transplantation experiments. Autograft and allograft behaviour in animals with BLAD was compared with the behaviour of simultaneously transplanted autografts and allografts in healthy controls. Allograft survival time was prolonged in three BLAD cattle (28, 30, and 72 days) compared to six healthy controls (12-14 days). When transplantations were repeated on one animal with BLAD using skin grafts from the same donor, accelerated rejection was observed (allograft survival time decreased from 72 days at primary to 35 days at secondary and to 21 days at tertiary transplantation), suggesting the development of immunological memory. Graft-infiltrating lymphocytes that were obtained from allograft biopsies during the period of rejection, were shown to be from recipient origin (beta 2-integrin negative). Our findings demonstrate that, although prolonged allograft survival is observed in cattle with BLAD, skin allografts are ultimately rejected.