Trypsin Inhibitor and Immunoglobulins in Porcine Colostrum

The specific trypsin inhibitor in porcine colostrum first described by Laskowski et al. (1957) is assumed to protect maternal antibodies in colostrum during absorption from the gut of the neonatal piglets (Baintner 1973). Investigations of Jensen & Pedersen have shown that the serum levels of IgG and IgA in newborn suckling piglets depend on both the immunoglobulin and the trypsin inhibitor levels in the colostrum of their mothers. Accordingly, the sow colostrum trypsin inhibitor (SCTI) is essential in order to ensure optimal systemic antibody protection to the newborn and young piglets. The secretory IgA in colostrum and milk, which gives local passive immunity to the gastro-intestinal tract of the piglets (Bourne 1973), is assumed in itself to be relatively resistant against proteolytic degradation (Tomasi & Bienenstock 1968).     

Maternal and neonatal somatomedin C/insulin-like growth factor-I (IGF-I) and IGF binding proteins during early lactation in the pig

RIA for insulin-like growth factor-I (IGF-I) was performed on Tris-neutralized, acid-ethanol extracts of porcine, bovine, ovine and human mammary secretions, and porcine maternal and neonatal sera. High levels (50-500 ng/ml) of immunoreactive IGF-I were present in the colostrum of all three animal species. IGF-I was also identified in porcine milk, though at levels 10- to 100-fold reduced relative to that in colostrum. Maternal (pig) sera was characterized by IGF-I concentrations intermediate between that in colostrum and that in milk. IGF-I levels were relatively low in serum of newborn pigs and exhibited an approximately 1.4-fold increase between Days 3 and 7 of postnatal life. Fractionation of pig colostrum in nondenaturing, gel-filtration columns demonstrated association of endogenous IGF-I with two prominent binding proteins (Mr's of 150,000 and 50,000 for the complexes). A third immunoreactive component was also observed to elute in the column void volume fractions (Mr greater than 158,000). The 150,000 and 50,000 Mr complexes were also present in serum obtained from sows at term. In contrast, IGF-I immunoreactivity in porcine milk was localized exclusively in the 150,000 Mr complex. Incubation of porcine colostrum and milk with 125I-IGF-I revealed a prominent, unoccupied IGF binding protein corresponding to that of the 150,000 Mr complex, whereas serum obtained from sows at term displayed both the 150,000 and 50,000 Mr unoccupied forms. Fractionation of (pooled) serum obtained from porcine neonates immediately at birth revealed a heterogeneous pattern of IGF-I immunoreactivity which included both the 150,000 and 50,000 Mr forms. Qualitative differences in this chromatographic pattern were apparent in serum at 6 hr postnatal and after ingestion of colostrum had occurred. The unoccupied IGF binding proteins in newborn pig serum were solely of the small size class. These results demonstrate that mammary secretion of IGF-I and its binding proteins are temporally regulated during the period immediately surrounding parturition. Physiologic alterations in the serum IGF-I profile during early postnatal life may reflect in part the uptake and/or response of the neonate to maternal IGF-I.     

Review: passive immunity in beef-suckler calves

Colostrum-derived passive immunity is central to the health, performance and welfare of neonatal beef-suckler calves, and economics of beef-farming enterprises. Compared to dairy calves, mainly Holstein-Friesian, there is much less research carried out on passive immunity and associated factors in beef calves. Thus, this review aimed to summarise and interpret published information and highlight areas requiring further research. The transfer of immunoglobulin G1 (IgG1) from blood to mammary secretions is greater for beef × dairy cows compared to most beef breed types. Considerable between-animal variance is evident in first-milking colostrum yield and immunoglobulin concentration of beef-suckler cow breed types. First-milking colostrum immunoglobulin concentrations are similar for within-quarter fractions and for the front and rear quarters of the udder. First-milking colostrum yield is higher for beef × dairy cows than beef × beef and purebred beef breeds, and higher for multiparous than primiparous cows, but generally colostrum immunoglobulin concentration is relatively similar for each of the respective categories. Consequently, colostrum immunoglobulin mass (volume × concentration) production in beef cows seems to be primarily limited by colostrum volume. The effect of maternal nutrition during late gestation on colostrum yield is not well documented; however, most studies provide evidence that colostrum immunoglobulin concentration is not adversely affected by under-nutrition. Factors that impinge upon the duration between birth and first suckling, including dam parity, udder and teat anatomy and especially dystocia, negatively impact on calf passive immunity. Colostrum immunoglobulin mass ingested relative to birth weight post-parturition is the most important variable determining calf passive immunity. Research indicates that feeding the beef calf a colostrum volume equivalent to 5% of birth weight shortly after parturition, with subsequent suckling of the dam (or a second feed) 6 to 8 h later, ensures adequate passive immunity, equivalent to a well-managed suckling situation. Within beef-suckler cow genotypes, calf passive immunity is similar for many common beef breeds, but is generally higher for calves from beef × dairy cows. Compared to older cows, calves from younger cows, especially primiparous animals, have lower serum immunoglobulin concentrations. Most studies have shown no adverse impact of maternal dietary restriction on calf passive immunity. The prevalence of failure of passive transfer (FPT) in beef calves varies considerably across studies depending on the test used, and what cut-off value is assumed or how it is classified. The accuracy and precision of methodologies used to determine immunoglobulin concentrations is concerning; caution is required in interpreting laboratory results regarding defining colostrum ‘quality’ and calf passive immune ‘status’. Further research is warranted on colostrum-related factors limiting passive immunity of beef calves, and on the validation of laboratory test cut-off points for determining FPT, based on their relationships with key health and performance measures.     

Molecular cloning and expression of the porcine high-affinity immunoglobulin G Fc receptor (FcγRI)

Receptors for the Fc region (FcγRs) of immunoglobulin G (IgG) play a crucial role in the immune system and host protection against infection. In this study, we describe the cloning, sequencing, and expression of the high-affinity IgG receptor from pig. By screening a translated Expressed Sequence Tags database with the human FcγRI (CD64) protein sequence, we identified a putative porcine homologue. Subsequent polymerase chain reaction amplification confirmed that the identified full-length cDNA was expressed in porcine cells. Rosetting analysis shows that COS-7 cells transfected with a plasmid containing the cloned cDNA were able to bind chicken erythrocytes sensitized with porcine IgG. Scatchard analysis indicated that monomeric IgG bound to transiently transfected cells with an affinity of approximately 4×10⁷ M⁻¹. The porcine FcγRI cDNA is 1,038 nucleotides long and is predicted to encode a 346-amino-acid transmembrane glycoprotein composed of three Ig-like domains, a transmembrane region, and a short cytoplasmic tail. The overall identity of the porcine FcγRI to its human and mouse counterparts at the level of the amino acid sequence was 75% and 57%, respectively. Identification of porcine FcγRI will aid in the understanding of the molecular basis of the porcine immune system and further studies of the receptor function.Gaiping Zhang and Songlin Qiao contributed equally to this study.The GenBank accession number of the nucleotide sequence reported here is DQ026063.     

Molecular characterization and association analysis of porcine interferon regulatory factor 1 gene

Interferon regulatory factor 1 (IRF1) is a member of IRF-family that was discovered to activate promoters in type I interferon (IFN) genes. It is shown to play functionally diverse role in the regulation of the immune system. In this report, the porcine IRF1 cDNA were cloned and a 7500 bp genomic DNA structure was identified. The putative IRF1 protein included 322 amino acids. Alignment and phylogenetic analysis of the predicted porcine IRF1 amino acids sequence with its homologies of other species show high identity (over 88%). Tissues expression of IRF1 mRNA was observed by RT-PCR, the results revealed IRF1 gene expressed widely in all analyzed tissues. Using the radiation hybrid panel, the porcine IRF1 gene was mapped to porcine chromosome 2 and closely linked to the locus IL4 (LOD = 7.09, 57cR). A SNP in exon2 of porcine IRF1 gene was demonstrated by sequencing and PCR–RFLP analysis. The further association analysis indicated that the SNP was significant associate with level of IFN-γ (day 20) in serum (P = 0.0001) and the ratio of IFN-γ to IL10 (day 20; day 35) in serum (P = 0.0165; P = 0.0095). The results suggested that the porcine IRF1 gene is strong candidate gene for these immune traits in pig.     

Seric and secretory antibodies reactive to α, β and γ intimins of Escherichia coli in healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to α, β and γ intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of α, β and γ intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.     

The Interactions of Porcine and Ovine,Serum and Colostral Immunoglobulins with Staphylococcal Protein A

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG₁, IgG₂, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG₁ from most ewes possessed a low affinity for Protein A whereas ovine IgG₂ generally possessed a high affinity; 100% of the IgG₂ in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.     

Evaluation of γ-rays irradiation of cows' colostrum for Brucella abortus, Escherichia coli K99, Salmonella dublin and Mycobacterium paratuberculosis decontamination

Deep-frozen cows' colostrum samples inoculated with high doses of either Brucella abortus strain 544, Escherichia coli K99, Salmonella dublin or Mycobacterium paratuberculosis were subjected to γ-irradiation for decontamination. The 10 kGy γ-irradiation which is commonly used for sterilization of foodstuffs and biological products proved to be efficient in eliminating the four organisms from colostrum without damage for anti-Bovine Herpes Virus 1 immunoglobulin specific activity.     

Inhibitory effects of human serum on human fetal skin fibroblast migration: Migration-inhibitory activity and substances in serum, and its age-related changes

Summary In order to clarify the environmental factors modulating cell migration, we investigated the effects of human serum on cell migration, and found that serum from adult donors strongly (by 48%) suppressed the migration of human fetal skin fibroblasts into a denuded area in a cell monolayer. Human serum from old donors inhibited cell migration more strongly than that from adult donors. Next, we investigated the properties of migration-inhibitory activity of human serum and serum proteins in order to identify migration-inhibitory substances. Human serum from adult donors strongly suppressed the migration of human fetal skin fibroblasts, although it stimulated cell proliferation more strongly than fetal bovine serum (FBS), indicating that the inhibitory effects of human serum on cell migration was not due to its toxic effects. The inhibition of cell migration by human serum was concentration dependent. It was demonsstrated that the inhibition did not depend on the inhibitory effects of human serum on collagen synthesis. The migration-inhibitory activity was seen in fractions over 100 kDa, as determined by an ultrafiltration membrane, and no inhibitory activity was observed in fractions under 100 kDa. On the other hand, it was not detected either in fractions over 100 kDa or under 100 kDa in FBS. Among the over 100 kDa human serum proteins examined, γ-globulin, α2-macroglobulin, and low density lipoprotein (LDL) suppressed fibroblast migration in a concentration-dependent manner. However, among the three, cell migration-inhibiting activity of γ-globulin almost disappeared when cell migration was conducted in 10% FBS-supplemented medium. These results indicated that α2-macroglobulin and LDL were candidate substances for cell migration-inhibiting activity in human serum.     

Quantitative detection of blood-brain barrier-associated enzymes in cultured endothelial cells of procine brain microvessels

Summary The present study deals with a rapid and convenient assay for blood-brain barrier (BBB)-associated enzymes, γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP), in cultured endothelial cells and other cells. These enzyme activities in cultured cells could be efficiently measured by direct incubation of each substrate in the culture plates without pretreatment of the cells. This new direct in situ-in plate assay was more rapid and convenient than conventional ex-plate assays, and these assays gave similar values for specific enzyme activities. γ-GTP and ALP activities could be detected by this in situ method in primary-cultured endothelial cells of porcine brain microvessels, but their levels were lower than those before culture. The degree of loss due to culture differed, between γ-GTP and ALP; a relatively large amount of ALP remained but the γ-GTP level decreased greatly In this direct in situ-in plate assay, cultured porcine aortic endothelial cells exhibited negligibly small activities for both enzymes, whereas cultured astroglial cells of neonatal porcine brain showed moderate γ-GTP activity and a trace of ALP activity. This direct in situ-in plate assay can be used for microculture and automatic measurement and offers a convenient means for studying the possible regulatory mechanisms of the expression of the BBB-associated enzymes.     

Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils

Summary. The effects of bovine colostrum, absorption of equine colostral immunoglobulins and age on phagocytic and serum opsonizing activity of nine clinically healthy foals were examined. Cells and serum were collected prior to suckling and at 7, 14 and 28 days of age. Seven foals had serum IgG concentrations >600mg/dl whereas two foals had <350mg of IgG/dl. Phagocytic and serum opsonic activity of eight clinically ill foals with <400mg of IgG/dl of serum were also examined before and after plasma transfusion. Phagocytic and serum opsonizing activities were evaluated by an assay for chemiluminescence (CL) after addition of opsonized streptococci. Results showed that bovine colostrum stimulated CL of foal neutrophils. Preliminary characterization of opsonins in bovine colostrum by ammonium sulphate fractionating and heat inactivation indicated that opsonins generating CL were mainly associated with immunoglobulin G. Chemiluminescence generated by foal neutrophils varied with age with foal neutrophils collected at day 14 producing more CL than adult neutrophils ( P <0.05). Foal serum opsonizing activity was similar to adult opsonizing activity if serum IgG concentrations were >600mg/dl but it was less if IgG concentration was <350mg/dl ( P <0.05). Chemiluminescence generated by foal and adult neutrophils was higher when post-transfusion foal serum was used as the source of opsonin than when pre-transfusion foal serum was used ( P <0.05). When adult serum was the opsonin, chemiluminescence of foal neutrophils collected before and after plasma transfusion did not differ. The increase in CL following plasma transfusion was probably due to an increase in serum opsonizing activity.     

Comparative studies of two types of bovine immunoglobulin G heavy chains

;Fingerprints' of bovine colostrum and serum immunoglobulin G1 heavy chains were extremely similar, but different from serum immunoglobin G2 heavy chains. Serum immunoglobulin G1 and immunoglobulin G2 heavy chains were treated with cyanogen bromide. The fractions from the C-terminal end of the heavy chains were isolated and the amino acid sequence of this fraction from immunoglobulin G2 was:His-Glx-Ala-Leu-His-Asx-His-Tyr-Met-Gln-Lys-Ser-Thr-Ser-Lys-Ser-Ala-GlyThe amino acid composition of this fraction from immunoglobulin G1 was the same except for the methionine, which in immunoglobulin G1 was replaced by threonine.     

Use of zinc ions for fractionation of pig γ-G-globulin and its structural subunits

The different behaviour of the two fractions of pig γ-G-globulin prepared by interaction with zinc ions during oxidative sulphitolysis is described. The γ-G-globulin fraction which is not precipitated by zinc ions is dissociated more readily, as seen from the finding that, unlike the other fraction, it contains practically no incompletely dissociated molecules. Fractions of the light chains, with different molecular weights, were also isolated from this fraction. A technique was elaborated for separation of the component with H antigenic specificity which is present in the light chain preparation. Detailed study of this component showed that it is probably part of the heavy chain. The origin and formation of the component is discussed.     

Properties of natural 19S antibodies in normal pig serum against the ΦX 174 and T2 phages

The physicochemical properties of natural phage-neutralizing antibodies were studied. Natural neutralizing antibodies against phages ΦX 174 and T 2 were found in the 19 S fraction isolated from normal pig serum by gel filtration on a Bio-Gel P-300 column. The 7 S fraction of normal pig serum possessed no neutralizing activity. The neutralizing activity of the 19 S fraction against phage ΦX 174 was not modified either by inactivation or by the addition of neutralizing cofactor; its activity against phage T 2 was lost by inactivation, but was restored by the addition of cofactor. The neutralizing activity of the 19 S fraction of normal pig serum was completely destroyed by 2-mercaptoethanol and was not restored by the subsequent addition of antibody cofactor. The results of attempts to release phage ΦX 174 from the neutralization complex with normal porcine serum 19 S macroglobulin antibody by the dilution method were the same as those of attempts to dissociate phage from the complex with 7 S type hyperimmune antibody. The virus particle was firmly and irreversibly adsorbed to both types of antibody and was not released by dilution. It is concluded from the results that neutralization of phage ΦX 174 by 19 S macroglobulin molecule of antibody is a simple, irreversible process, for which the thermolabile nonspecific serum components are not required.     

Contribution to the structural characterization of gamma globulin from pig colostrum

From the results obtained in the present work it is concluded that gamma globulin of the 7 S type (γG) which represents the main immunoglobulin component of pig colostrum, differs from serum γG globulin by the presence of another type of polypeptide chain, designed L₂; the latter was detected after S-sulfonation in starch gel electrophoresis as the fastest moving component and its immunoelectrophoretic pattern shows the presence of two precipitating components. Preparation of soluble heavy and light chains enabled us to study them imunochemically. The heavy chain is represented by two zones; the fainter one was not detected in comparative analysis of the heavy chain of serum γG globulin. The L₁ chain of colostral gamma globulin and the light chain of serum gamma globulin seem to contain three precipitating components, one of which appears due to aging of the chain solution in the presence of glycine. The material from colostrum seems to contain a larger amount of this component than serum. Further it was shown that the component arising in this way is antigenically closely related to the heavy chain.     

Studies on Immunoglobulins and Trypsin Inhibitor in Colostrum and Milk from Sows and in Serum of Their Piglets

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Metabolic correlates of the circadian pattern of suckling-associated arousal in young rabbits

Young rabbits (Oryctolagus cuniculus) are only nursed for 3–5 min every 24 h. They show a circadian increase in activity in anticipation of this, which is entrained by suckling. Our aim was to determine whether serum and liver metabolites show diurnal fluctuations which could act to regulate this circadian pattern. Stomach weight, liver glycogen and serum metabolites were measured every 3 h in 7- to 8-day-old pups when normally nursed (up to 24 h after suckling) and fasted (up to 48 h after suckling). The results suggest: Accepted: 9 October 1999     

Substrate contribution on free radical scavenging capacity of carotenoid extracts produced from <Emphasis Type="Italic">Blakeslea trispora</Emphasis> cultures

Blakeslea trispora produces carotenoids mixtures consisting mainly of lycopene, γ-carotene and β-carotene, together with trace amounts of other carotenoid precursors. The yield of these carotenoids and their composition are greatly affected by culture substrate. The scavenging capacity of carotenoids extract from cultures of B. trispora growing in various substrates was estimated using the 2,2-diphenyl-1-picrylhydrazyl method. Fractions enriched in β-carotene, γ-carotene and lycopene, obtained after column chromatography in alumina basic II, were also examined. Substrates containing starch and oils mixture, Ni²⁺, and that with pantothenic acid presented higher antioxidant activity. An increase in the antioxidant activity of the crude carotenoid extract compared to that of the isolated fractions enriched in β-carotene, γ-carotene and lycopene respectively, observed in most samples, indicated a possible synergistic effect. The results are of interest and by expanding this study to more substrates and other microorganisms- producing antioxidants, a formulation of extract with high free radical scavenging potential could be produced.     

Human colostral whey M-1 glycoproteins and their L. bifidus var. Penn. growth promoting activities

A total of seven M-1 glycoprotein fractions were isolated from human colostrum whey and all showed L. bifidus var. Penn. growth promoting activities using well defined chemical medium. The growth promoting activity of these fractions was parallel to their total carbohydrate content. On the other hand, bovine colostrum M-1 glycoprotein had a relatively low growth promoting activity, whereas human serum orosomucoid had none.