Assessment of Antibody‐independent Cellular Cytotoxicity (AICC) of Porcine Neutrophilic Granulocytes by Quantitative Flow Cytometry. Lack of Modulation by Larval Products of Oesophagostomum dentatum

Abstract

After infection of pigs by the larvae of Oesophagostomum dentatum, granulomas are formed around the third-stage larvae in the submucosa of the gut which contain a considerable number of neutrophils. This has no obvious impact on the larvae, which develop to fourth-stage larvae within these granulomas. We therefore asked, whether the products of O. dentatum larvae modulate the functional capacity of porcine neutrophils. The antibody-independent cellular cytotoxicity (AICC) was chosen as a model system. This assay was developed for the pig and quantified using flow cytometry. Bovine lymphoblastoid cells (cell line Anna TA1) served as targets. The measurement of cytotoxicity was based on the determination of absolute numbers of vital target cells. This procedure proved to be reliable and required no additional labelling of target and/or effector cells. Porcine neutrophils, when stimulated with phorbol 12-myristate 13-acetate (PMA; > or = 10 nmol/l), killed target cells at effector: target ratios between 1:1 and 9:1. AICC was not demonstrable after 4 h but could be observed between 16 h and 20 h after in vitro co-culture. Killing of targets required close physical contact between effector and targets, since supernatants of PMA-stimulated polymorphonuclear cells were not able to lyse the target cells. Homogenates of third- and fourth-stage larvae of O. dentatum did not affect the vitality of porcine granulocytes or target cells in vitro, nor did they modulate the AICC capacity of porcine granulocytes.

Flow Cytometric Testing of Immunological Effects of a Phytomedicinal Combination (EquiMun) and its Compounds on Bovine Leucocytes

“…We have chosen flow cytometric methods because of their sensitivity in the detection of even subtle effects on cells. This has been shown by our group in various other studies where cellular responses and function of porcine, bovine and piscine leucocytes have been studied in response to various stimuli invitro as well as ex vivo (Scharsack et al, 2000; Schuberth et al, 2000a, b).…”

“…Bovine lymphoblastoid cells (‘Anna TA1’, Schuberth et al, 2000a) were used as target cells (T). Targets were washed twice in PBS (80 g , 8 min) and adjusted to 1 × 10 6 cells/ml culture medium.…”

Histopathological changes during experimental infections of calves with Cooperia punctata

“…5), demonstrating the destruction of worms by the mobilized cell contingent. The presence of microcysts, although not demonstrated in other studies involving the genus Cooperia (Ransom, 1920;Hung, 1926;Bailey, 1949;Coop et al, 1979;Armour et al, 1987;Oliveira, 2001), was reported by Schuberth et al (2000) in experimental infections of swine with Oesophagostomum dentatum, and the formation of these structures was related to the mobilization of neutrophils and eosinophils. However, in contrast to the present study, no destruction of worms by the cell types in question was observed, and the larvae survived and developed inside the microcysts.…”