I/'eterinary Immunology and Immunopathology, 29 ( 1991 ) 127-138

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Elsevier Science Publishers B.V., Amsterdam

Serum opsonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake W.E. Bernadina a, M.A.W. van Leeuwen b, W.M.L. Hendrikx b and E.J. Ruitenberg ~ Departments ofaImmunolog~, and bHelminthology and Entomology of the Division of lnfectious Diseases and Immunology, Faculty of FeterinaryMedicine, Falelaan 1, 3584 CL Utrecht, Netherlands (Accepted 20 August 1990)

ABSTRACT Bernadina, W.E., van Leeuwen, M.A.W., Hendrikx, W.M.L. and Ruitenberg, E.J., 1991. Serum o p sonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake. Vet. Immunol. Immunopathol., 29: 127-138. Neutrophil (PMN) counts, immune complex (IC) uptake by PMN, and serum opsonising activity for promoting yeast uptake were used to evaluate infection clearing capacity in 16 lambs prior to colostrum feeding (two lambs fed bovine colostrum, 14 suckled lambs) and at 2 days of age. At 2 days of age lambs had more circulating PMN than they had prior to colostrum uptake (P< 0.01 ). i~,~lnetr,,m

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though at Day 2 the percent phagocytosis was significantly lower (32.2%) than for adult controls (90%). Yeast opsonophagocytosis was greater when 24-36 h post-feeding serum was the source of opsonin than when pre-feeding serum was used (P< 0.001 ). When adult serum was the opsonin, yeast opsonophagocytosis was approximately twice the phagocytosis mediated by 24-36 h post-feeding serum. The peripheral neutrocytosis and the enhancement of opsonophagocytosis generated by absorption of either ovine or bovine colostrum did not differ. The results of this study suggest that the parameters evaluated may be used for indicating the presence (or absence) of passively acquired protective immunity. ABBREVIATIONS C, complement; FITC, fluorescein isothiocyanate; IC, immune complex; PBS, phosphate-buffered saline; PMN, polymorphonuclear neutrophil.

INTRODUCTION

Peripheral neutrocytosis and enhancement of serum opsonic activity and phagocytic efficiency are prominent features in neonates, which when fed co0165-2427/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

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lostrum, subsequently develop resistance to infections (Tizard, 1987). It is also well established that absorbed colostral immunoglobulins (Ig) function to induce immunity in lambs (Campbell et al., 1977; Awad-Masalmeh and Willinger, 1985; Mellor, 1985; Mellor and Murray, 1986). Hence, to analyse the immune status of newborn farm animals, including lambs, the passive serum Ig concentration may be determined (Reid, 1972; Halliday, 1974; Tizard, 1987; Caldow et al., 1988 ). There is accumulating evidence from human studies to indicate a lack of relation between levels of serum opsonins and effective clearing of infections. On one hand, normal serum levels of complement (C) or antigen-specific Ig may often mediate defective opsonophagocytosis (Levinsky et al., 1978; Baker et al., 1986; Turner et al., 1986). On the other hand, normal opsonophagocytosis may be found in conjunction with low serum levels of either C or specific Ig (Kerr et al., 1983; Musher et al., 1986). Together, these data, when extrapolated to sheep, would suggest that a neonatal lamb's passive serum Ig concentration is a dubious measure of protection. Consequently, as a prelude to the development of a more reliable measure, peripheral neutrophil numbers, phagocytic and serum opsonic activity of colostrum-deprived newborn lambs were compared with those of healthy lambs 24-36 h after first uptake of either cow or sheep colostrum. MATERIALS AND METHODS

Experimental animals Sixteen Texei lambs were used. At birth, lambs had their navels treated with iodine and were left with their dam. Eight lambs (Lambs 150-157) were allowed immediate natural suckling whereas the other eight lambs ( 142-149) were bled before suckling was allowed. Two lambs ( 148, 149) had mothers who resisted suckling and these lambs were each bottle-fed about 21 of bovine colostrum over three feeds per day. Blood samples for serum and polymorphonuclear neutrophil (PMN) separation were obtained by jugular venepuncture (B-D vacutainers) from the lambs prior to colostrum feeding and at 1-2 days of age (hereafter designated pre-feeding and post-feeding lambs, respectively). Blood was also obtained from three healthy adult sheep (normal controls, Lambs 45, 48, 207 ).

Buffers Saline was 0°9% NaCI. Phosphate-buffered saline (PBS), pH 7.4. Heparin (Thromboliquine: 5000 IU ml -~, Organon, Holland), 2 ml/l was added to PBS to make PBS-hep.

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Preparation of immune complex (IC)-containing sera Soluble Haemonchus contortus and Trichostrongylus colibriformis immune complexes (Hc-IC and Tcol-IC, respectively) were prepared as follows. Serum was obtained from sheep hyperimmunised against/-/_ contortus (HiS.Hc) or T. colibriformis (HIS.Tcol). The His.Hc and HIS.Tcol sera were decomplemented and incubated ( 1 h at 37 ° C with gentle shaking) with soluble Hc and Tcol antigens, respectively (fresh L3-stage nematodes, pulverised after freezing in liquid nitrogen, centrifuged ( 50 000 ×g, I h) and passed through a 0.45 gm filter) at slight antigen excess. After another filter-sterilisation the ICcontaining sera Were stored at 4 ° C to be used within 3 h of being prepared.

Yeast preparations Commercially obtained baker's yeast was resuspended in saline, filtered through gauze mesh, boiled for 30 min and stored for 2 h at 4 ° C. The yeast was then heated to 100°C for 60 rain, washed extensively (5 × ), counted (5 × ) in a haemocytometer, and adjusted to a concentration of 1 × 108 particles/ml in saline. This stock preparation was stored at 4 ° C. Yeast particles were labeled with fluorescein-isothiocyanate (FITC, Isomer 1; Sigma, U.S.A. ) by gently stirring 25 ml of yeast particles ( 108)/ml in 0.5 M sodium bicarbonate buffer (pH 9.5 ) with 10 mg FITC dissolved in 25 mi acetone for 2 h at 20 ° C. The yeast particles were washed in saline (2 × ) and PBS (3 × ), resuspended in PBS, counted (5 × ) and adjusted to a final concentration of 1 × 108 particles/ml in PBS.

Serum samples Sera from lambs and normal controls (hereafter designated pre-feeding, post-feeding and adult sera) were separated within 2 h of collection, filtersterilised (0.45/zm) and used either fresh or stored at - 2 0 ° C for short periods (up to 2 days) before assay.

Separation of PMN PMN were prepared from lamb (8-9 ml) and control sheep (20 ml) whole blood using the Ficoll-Isopaque separation technique. Briefly, the bulk of the erythrocytes plus PMN were separated from the mononuclear leucocytes by layering blood, diluted 1 : 1 with sterile PBS, onto Ficoll-Isopaque (d = 1.078 ) and centrifuged at 2000 r.p.m, for 45 min, 20 ° C. The Ficoll-plasma interfaces were aspirated, washed (2 × ) in PBS, and cell pellets in 5% (v/v ) autologous plasma-supplemented PBS were stored on ice. The pellets containing the PMN were recovered and the erythrocytes were removed by the addition of sterile deionized water (20 s, 20°C) followed by sequential washing with doublestrength PBS and PBS ( 1200 r.p.m., 10 rain, 4°C). The purity of PMN, as assessed by staining (May-Grunwald/Giemsa), was 93-96%, and the viability, determined by trypan exclusion, was greater than 95%. The white cells

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present in the Ficoll-plasma interfaces were at least 97% mononuclear ( 1-12% monocytes), and 1-3% PMN. PMN suspensions were counted (4 × ) in a haemocytometer, adjusted to 1 × 107 cells/ml PBS and stored on ice for immediate use.

Complement inactivation Complement activity in sera was removed by either heating (30 rain at 56°C) orthe addition of EDTA (50 gl, 0.02 M, pH 7) to 250 #1 serum and incubation at room temperature for 2 h).

lg-depleted serum Ig was removed from serum samples by adsorption to Sepharose 4B CNBractivated beads (Pharmacia, Sweden) containing mono-specific antibodies against sheep IgGl, IgG2, IgM and IgA (prepared by procedures essentially as described by Verdouw-Chamaloun et al., 1977).

Opsonophagocytic assays In these assays suspensions were prepared in PBS, unless stated otherwise. All reaction mixtures were left with gentle shaking at 37°C for the incubation period after which time reactions were terminated by the addition of ice-cold PBS and immersing the reaction tubes in crushed ice for 5 min. In a pilot study, the following methods of assaying yeast opsonophagocytic capacities of human PMN were shown to be similar to sheep PMN without major mndificatian~_

Effect of colostrum uptake on phagocytic capacity of lamb PMN A total of 100 gl IC-serum or control medium and 100 gl lamb PMN (106/ml) were mixed together and left for 45 rain. After two buffer washes ( 1000 r.p.m., 5 rain, 4 °C), the pellet was resuspended in 250 gl PBS. A sample was removed, spun onto a microscope slide with a cytocentrifuge (500 r.p.m., 3 rain), airdried and fixed in acetone for 10 min. The slide was washed and 1:40 diluted FITC-conjugated rabbit anti-sheep IgG (H + L) (Miles, Israel) was applied for 30 min. The slide was rinsed, mounted and scored for antibody (IgG)-mediated phagocytosis by fluorescence microscopy (at least 100, but usually 200-250 PMN examined per slide). All slides were examined twice (in 3 days) by one investigator who was blinded as to the slide's code (the two scores per slide were consistently shown to differ less than 10%). The data are expressed as percent phagocytosis, i.e. the percentage of PMN that had ingested five or more fluorescent particles. (In nearly all lamb PMN preparations, 6-10% of the PMN contained less than five, and mostly two fluorescent particles. )

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Incubation times for distinguishing normal from deficient lamb sera The Levinsky et al. ( 1978 ) protocol for yeast uptake was followed but here uptake was determined by countings from a haemocytometer. Briefly, yeast particles ( 5 × ! 06 in saline) were incubated for 15, 30 and 45 min in the presence of adult PMN ( 106 in saline) and either 5 or 0% ( v / v ) serum in a total volume of 200/tl saline. After each incubation time, five samples were removed for determining percent yeast uptake (see below). Assessment of normal values in neonatal and adult sera The slide opsonisation test used both standard yeast particles and FITClabelled yeast particles, and employed a fluorescence quenching technique (Bjerknes and Bassoe, 1984) to distinguish between PMN-attached particles and ingested ones. The tests were carried out essentially as described by Miller et al. (1968). Briefly, 100 gl yeast particles (5 × 107/ml), 10/d serum+ 90/d PBS (or 100 gl PBS only) were added to 200 gl sheep PMN ( 5 × 106/ml ) and left for periods of 15 and 30 rain. Following washes with buffer ( 1000 r.p.m., 5 min) to remove all remaining serum, the pellet was resuspended in: ( 1 ) 250 #1 PBS, after which triplicate samples were air-dried onto microscope slides, stained (May-Grunwald/Giemsa), and examined for yeast uptake under oil immersion ( × 100 magnification); (2) 250 gl PBS+ 250 gl fluorescence quenching solution (trypan blue, 1 m g / m l ) a n d stored at 10°C for 20 rain, and then centrifuged at 1000 r.p.m, for 5 min. The resulting pellet was resuspended in 50/zl PBS + 50 gl trypan blue solution after which triplicate samples were dropped onto microscope slides and covered by glass cover slips. preparations that remained viable for at least 2 days at 4°C. The slides were examined for yeast uptake using a combination of light (phase-contrast) and fluorescence microscopy.

Parameters of yeast opsonophagocytosis The percentage uptake of yeast was calculated from the formula: percent yeast uptake= (Ya--Yu):YaM 100%, where Ya is number of yeast particles added to the reaction mixture and Yu is the number of yeast particles left unphagocytosed. (Ya and Yu were average scores of five counts in a haemocytometer. ) The percent phagocytosis in slide opsonisation experiments was calculated from the equation: percent phagocytosis- (Fm: Mm) × 100 where Fm is the calculated mean number of yeasts per PMN (counting at least 100 PMNs) and Mm is the maximum mean number of yeasts per PMN attainable ( - f i v e yeasts per PMN). The results represent mean scores of at least three determinations.

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Statistics Where appropriate, Student's t-test for paired/unpaired determinations was used to assess the significance of differences between mean values. P values less than 0.05 were considered significant. RESULTS

Colostrum-induced neutrocytosis and PMN recovery At 2 days of age lambs had more (P

Serum opsonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake.

Neutrophil (PMN) counts, immune complex (IC) uptake by PMN, and serum opsonising activity for promoting yeast uptake were used to evaluate infection c...
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