Purification of Ovine Neutrophils and Eosinophils: Anaplasma phagocytophilum affects Neutrophil Density

Woldehiwet, Z.; Scaife, Helena; Hart, C. A.; Edwards, Steven W.

doi:10.1053/jcpa.2002.0633

Cited by 17 publications

(13 citation statements)

References 11 publications

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“…Our efforts to isolate eosinophils by the immunomagnetic method [5] have also failed probably due to lack of recognition of sheep neutrophils by human anti-CD16 antibody. However, the 92-99% neutrophils isolated with this technique was close to those found by other authors [15] and the mononuclear cell suspensions were almost free of granulocytes. The motility of larvae incubated for 18 h with eosinophil-enriched suspensions was drastically reduced while the effects of neutrophils and mononuclear cells were much lower.…”

Section: Discussionsupporting

confidence: 89%

“…In spite of repeated attempts to optimise the isolation method, we did not attain previously reported levels of purity [15] probably because of the difference in the sheep breed used. Our efforts to isolate eosinophils by the immunomagnetic method [5] have also failed probably due to lack of recognition of sheep neutrophils by human anti-CD16 antibody.…”

Section: Discussioncontrasting

confidence: 56%

“…The different types of blood leucocytes were isolated using the method adapted from techniques developed by Woldehiwet et al [15]. Briefly, 30 mL of blood was taken from each Black Belly sheep, on day 15 post infection, in EDTA-coated tubes.…”

Section: Methodsmentioning

confidence: 99%

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In vitro pre-exposure ofHaemonchus contortusL3 to blood eosinophils reduces their establishment potential in sheep

et al. 2007

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-Different authors have reported that eosinophils are capable of immobilising infective larvae of different species of nematodes in vitro. However, classifying larvae as mobile or immobile is so subjective that it does not always mean all apparently immobile larvae are dead or those that are mobile are capable of surviving further immune responses if administered to their natural hosts. The objective of this experimental study was therefore to substantiate the role of eosinophils in the killing of Haemonchus contortus infective larvae by comparing the infectivity in sheep of larvae that had been incubated with eosinophil-enriched cell suspensions with control larvae. Since it was not possible to isolate pure eosinophils from sheep blood, we were compelled to evaluate the effects of other blood cells contaminating our eosinophil-enriched suspensions. Although eosinophils and neutrophils were the only cells found adherent to H. contortus infective larvae in vitro, induced eosinophils in the presence of immune serum were primarily responsible for the drastic reduction in larval motility compared to the minor effects of neutrophils and mononuclear cells. Corresponding reductions in faecal egg count and worm numbers were observed when the incubated larvae were transferred intra-abomasally to sheep. Interestingly, the proportion of larvae that failed to establish was much higher following incubation with induced eosinophils compared with other cells or with immune serum alone. Although this study did not address the in vivo role of eosinophils in sheep, the results strongly indicate that sheep blood eosinophils have a larval killing potential in vitro, and a larval mobility test alone may not fully explain the level of damage inflicted on the larvae.Haemonchus contortus / eosinophils / in vitro / intra-abomasal / sheep

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Section: Discussionsupporting

confidence: 89%

Section: Discussioncontrasting

confidence: 56%

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In vitro pre-exposure ofHaemonchus contortusL3 to blood eosinophils reduces their establishment potential in sheep

et al. 2007

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“…with a British (Old Sourhope) strain of A. phagocytophilum by using 1 ml of a 10% dilution of blood stabilate with 10% dimethyl sulfoxide (20,25,26).…”

Section: Methodsmentioning

confidence: 99%

Serologic Cross-Reactivity between Anaplasma marginale and Anaplasma phagocytophilum

Dreher

Fuente

Hofmann‐Lehmann

et al. 2005

Clin Vaccine Immunol

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In the context of a serosurvey conducted on the Anaplasma marginale prevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginale and A. phagocytophilum might exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilum not only develop antibodies to A. phagocytophilum (detected by immunofluorescent-antibody assay) but also to A. marginale (detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginale also developed antibodies to A. phagocytophilum using the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginale and one of A. phagocytophilum supported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginale and A. phagocytophilum infection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully.

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“…Eosinophils were collected from 45%/58.5% interface of the discontinuous Percoll gradients (GE Healthcare). Where PB eosinophils were required at higher levels of purity, eosinophils were purified according to the method of Woldehiwet [15], which included an isotonic lysis step. Eosinophils were obtained from the 65/70% interface of the discontinuous Percoll gradients and were >90% pure with no more than 8% contaminating neutrophils.…”

Section: Collection and Purification Of Peripheral Blood (Pb) Eosinopmentioning

confidence: 99%

Functional characterization of an eosinophil-specific galectin, ovine galectin-14

et al. 2008

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Across mammalian species, human galectin-10 and ovine galectin-14 are unique in their expression in eosinophils and their release into lung and gastrointestinal tissues following allergen or parasite challenge. Recombinant galectin-14 is active in carbohydrate binding assays and has been used in this study to unravel the function of this major eosinophil constituent. In vitro cultures revealed that galectin-14 is spontaneously released by eosinophils isolated from allergen-stimulated mammary gland lavage, but not by resting peripheral blood eosinophils. Galectin-14 secretion from peripheral blood eosinophils can be induced by the same stimuli that induce eosinophil degranulation. Flow cytometric analysis showed that recombinant galectin-14 can bind in vitro to eosinophils, neutrophils and activated lymphocytes. Glycan array screening indicated that galectin-14 recognizes terminal N-acetyllactosamine residues which can be modified with alpha1-2-fucosylation and, uniquely for a galectin, prefers alpha2- over alpha2-sialylation. Galectin-14 showed the greatest affinity for lacto-N-neotetraose, an immunomodulatory oligosaccharide expressed by helminths. Galectin-14 binds specifically to laminin in vitro, and to mucus and mucus producing cells on lung and intestinal tissue sections. In vivo, galectin-14 is abundantly present in mucus scrapings collected from either lungs or gastrointestinal tract following allergen or parasite challenge, respectively. These results suggest that in vivo secretion of eosinophil galectins may be specifically induced at epithelial surfaces after recruitment of eosinophils by allergic stimuli, and that eosinophil galectins may be involved in promoting adhesion and changing mucus properties during parasite infection and allergies.

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Purification of Ovine Neutrophils and Eosinophils: Anaplasma phagocytophilum affects Neutrophil Density

Cited by 17 publications

References 11 publications

In vitro pre-exposure ofHaemonchus contortusL3 to blood eosinophils reduces their establishment potential in sheep

In vitro pre-exposure ofHaemonchus contortusL3 to blood eosinophils reduces their establishment potential in sheep

Serologic Cross-Reactivity between Anaplasma marginale and Anaplasma phagocytophilum

Functional characterization of an eosinophil-specific galectin, ovine galectin-14

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