The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Microsatellite diversity, population subdivision and gene flow in the Lipizzan horse.

Blood samples of 561 Lipizzan horses from subpopulations (studs) of seven European countries representing a large fraction of the breed's population were used to examine the genetic diversity, population subdivision and gene flow in the breed. DNA analysis based on 18 microsatellite loci revealed that genetic diversity (observed heterozygosity = 0.663, gene diversity = 0.675 and the mean number of alleles = 7.056) in the Lipizzan horse is similar to other horse breeds as well as to other domestic animal species. The genetic differentiation between Lipizzan horses from different studs, although moderate, was apparent (pairwise F(ST) coefficients ranged from 0.021 to 0.080). Complementary findings explaining the genetic relationship among studs were revealed by genetic distance and principal component analysis. One genetic cluster consisted of the subpopulations of Austria, Italy and Slovenia, which represent the classical pool of Lipizzan horse breeding. A second cluster was formed by the Croatian, Hungarian and Slovakian subpopulations. The Romanian subpopulation formed a separate unit. The largest genetic differentiation was found between the Romanian and Italian subpopulation. Genetic results are consistent with the known breeding history of the Lipizzan horse. Correct stud assignment was obtained for 80.9% and 92.1% of Lipizzan horses depending on the inclusion or exclusion of migrant horses, respectively. The results of the present study will be useful for the development of breeding strategies, which consider classical horse breeding as well as recent achievements of population and conservation genetics.

The effect of different quantities and compositions of pelleted diets on immune response of mares during the production of anti-tetanus sera.

Research was carried out into the effect that different quantities and compositions of concentrated portions of meal had on certain haematological properties and on the immune response of mares in the course of hyper-immune antitetanus sera production. The experiment involved 24 Nonius and Lipizzaner cross-bred mares divided into two groups of 12 animals each, a control group and a trial group. The experiment lasted 12 months, with haematological and immunological tests being carried out every 30 days. During the course of the experiment each mare was subjected to 11 immunisation cycles, and in that period no mare fell ill. Leukocyte and haematocrit counts revealed no differences between the control and the test groups throughout the entire duration of the experiment. Following the second immunisation cycle the leukocyte count increased in both groups of mares, remaining significantly higher than the normal physiological value in horses throughout the experiment. The concentrated portion of meal, differing both in quality and quantity, had no influence on leucocyte count and haematocrit level in the mares throughout the experiment. However, the immunodiffusion titre in the test group did show a slightly higher value of ID-titre than in the control group, indicating that the quantitatively and qualitatively differing portions of meal had a more positive effect on immune response and anti-tetanus anti-body titre in the test group than it did in the control group.

Influence of environmental and genetic factors on allergen-specific immunoglobulin-E levels in sera from Lipizzan horses.

To investigate whether allergen-specific IgE production is influenced by environmental and genetic factors, IgE levels against 2 mould extracts (Alternaria alternata [Alt a] and Aspergillus fumigatus [Asp f]) and against recombinant (r) rAlt a 1, rAsp f 7 and rAsp f 8 were determined by ELISA in sera from 448 Lipizzan horses living in 6 studfarms. Statistical evaluation showed a significant effect of studfarm-specific environment on IgE levels against the different allergens, but genetic factors also influenced allergen-specific IgE production: an heritability of 0.33 was found for IgE levels against the 2 mould extracts and of 0.21 for rAsp f 8-specific IgE. Heritability estimates for rAlt a 1- and rAsp f 7-specific IgE were negligible. Investigations for a possible association between Major Histocompatibility Complex (MHC) class I antigens and specific IgE levels were carried out. The most consistent significant association was found between the equine leucocyte antigen (ELA) A8 and undetectable IgE titres against rAsp f 7 and rAsp f 8. Significant ELA associations were also demonstrated between ELA A1 and higher specific IgE levels, between ELA A14 and lower IgE levels against the mould extracts and in one studfarm between ELA Be27 and lower Aspergillus-specific IgE levels.

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed.

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.

The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences.

An abundant cytoplasmic 43-kDa protein from Mycoplasma synoviae, a major pathogen from poultry, was identified as elongation factor Tu. The N-terminal amino acid sequence (AKLDFDRSKEHVNVGTIGHV) has 90% identity with the sequence of the Mycoplasma hominis elongation factor Tu protein. Monoclonal antibodies reacting with the M. synoviae elongation factor Tu protein also reacted with 43-kDa proteins from the avian Mycoplasma species Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma pullorum, Mycoplasma cloacale, Mycoplasma iners and Mycoplasma meleagridis, but not with the proteins from Mycoplasma gallisepticum, Mycoplasma imitans or Mycoplasma iowae. In addition, two groups of phase variable integral membrane proteins, pMSA and pMSB, associated with hemadherence and pathogenicity of M. synoviae strains AAY-4 and ULB925 were identified. The cleavage of a larger hemagglutinating protein encoded by a gene homologous to the vlhA gene of M. synoviae generates pMSB1 and pMSA1 proteins defined by mAb 125 and by hemagglutination inhibiting mAb 3E10, respectively. The N-terminal amino acid sequences of pMSA proteins (SENKLI ... and SENETQ ...) probably indicate the cleavage site of the M. synoviae strain ULB 925 hemagglutinin.

The hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens more frequently than does the hemagglutination-negative phenotype.

Inoculation with hemagglutination-positive (HA+) cultures of Mycoplasma synoviae AAY-4 induced acute synovitis significantly more frequently (P = 0.001) in chicken tibiotarsal-tarsometatarsal joints than did inoculation with HA-negative (HA-) cultures derived from the same clone of AAY-4. Immunoblotting analyses showed that HA+ cultures abundantly expressed two phase-variable hemadherence-associated surface membrane proteins of 53 kDa and 48 to 50 kDa defined by monoclonal antibodies. HA- cultures lacked the 53-kDa proteins and synthesized truncated 27- to 30-kDa forms of the 48- to 50-kDa proteins. Inoculation of cyclosporin A (CsA) into infected joints significantly decreased the frequency of acute synovitis (P = 0.001). Moreover, repeated intra-articular inoculation of CsA (three doses of 1 mg at 2-day intervals) significantly reduced the local antibody response to M. synoviae in the joints treated with CsA.

[The absorption of colostral immunoglobulins in newborn piglets. III. Influence of the duration of colostrum administration]. / Untersuchungen über die Absorption der kolostralen Immunglobuline bei neugeborenen Ferkeln. III. Mitteilung: Einfluss der Verabreichungsdauer der Kolostralmilch.

The gastrointestinal tract of newborn piglets is permeable for intact immunoglobulins ingested with the colostrum. The duration of this passage was investigated by administering hourly rations of 25 ml of either porcine or bovine colostrum for 6, 12, 18 or 24 hrs after birth. The plasma concentrations of the subclasses porcine IgG, IgM and IgA or bovine IgG1, IgG2, IgM and IgA were determined at 12, 18 and 24 hrs after birth and on days 3 and 6. Feeding periods of 6 hrs resulted in plasma Ig levels of the same order of magnitude as observed in natural rearing. These levels were not substantially increased after prolonged feeding. The 6% gain from 6 to 12 feedings seen with porcine colostrum as compared with a gain of 24% for bovine colostrum points at an earlier closure of the intestinal wall for the species-specific proteins. There was no further increase of Ig permeation after 12 hourly feedings. Growth performances and losses were identical in all groups.

[The absorption of colostral immunoglobulins in newborn piglets. II. Effect of water or glucose solutions on the permeability of the newborn intestine]. / Untersuchungen über die Absorption der kolostralen Immunoglobuline bei neugeborenen Ferkeln. II. Mitteilung: Einfluss von Wasser oder von Glukoselösung auf die Durchlässigkeit des neugeborenendarmes.

The extent and the duration of the passage of intact proteins from the intestinal lumen into the bloodstream of newborn piglets was investigated. A total of 48 piglets from 4 litters were used in groups of 6 animals per experiment. The piglets were removed from the sow immediately after birth and placed in individual boxes of an automatic feeding device. Liquids were offered in trays, access to which was controlled by electronically operated gates. Volumes of 25 ml were allotted in hourly intervals. Porcine colostrum (6 x 25 ml) or bovine colostrum (24 x 25 ml) were either offered immediately, or after a 24 h fasting period without or with access to tap water. Two groups received 24 hourly rations of a 13 % glucose solution prior to the onset of feeding with porcine and bovine colostrum, respectively. The plasma levels of immunoglobulins were assessed by radial diffusion in blood samples drawn from a subcutaneous abdominal vein. The passage of intact proteins ceases between 12-18 hrs after the onset of feeding. Both the time course of the passage and the maximal levels achieved are unaffected after previous fasting or after tap water allowances only for 24 hrs. Previously fed glucose solution--in contrast--closes the gut barrier to subsequently ingested proteins. There was no difference in the postexperimental weight developments between groups.

[The absorption of colostral immunoglobulins in newborn piglets. I. Effect of time from birth to the first feeding]. / Untersuchungen über die Absorption der kolostralen Immunglobuline bei neugeborenen Ferkeln. I. Mitteilung: Einfluss der Zeit von der Geburt bis zur ersten Nahrungsaufnahme.

The effects of a delayed onset of feeding on the absorption of intact immunoglobulins from the small intestines was investigated in newborn piglets by using an automatic device ("artificial sow"). Fasting periods extended to a maximum of 24 hours and were followed by 12 hourly allotments of 25 ml of sow colostrum. The concentrations of immunoglobulins G, A and M were analyzed in plasma samples drawn before and after the onset of feeding. The capacity for Ig-absorption was not impaired by the fasting. The same plasma levels of 12 and 18 hours after the onset of feeding were obtained in piglets fed immediately after birth as observed with individual variations in all experimental groups, for which we could find no explanation. Our results indicate, that the absorptive ability of the intestinal epithelia for immunoglobulins is not timed from birth but rather from the onset of feeding.

Maternal-neonatal immunoregulation: suppression of de novo synthesis of IgG and IgA, but not IgM, in neonatal pigs by bovine colostrum, is lost upon storage.

Fifty-four neonatal pigs were allotted to 4 groups and reared in an electrically controlled automatic feeding device (autosow). Each group was reared on a different pool of bovine colostrum: fresh, stored 1 month, stored 6 months, and stored 8 years. Bovine and porcine immunoglobulins in the sera of these pigs, and in a group of conventionally reared pigs, were measured periodically during the first 42 days after birth. The maximal concentration of absorbed bovine immunoglobulin was reached between 12 and 18 hours and equaled or exceeded the amount of porcine immunoglobulin absorbed by the conventionally reared pigs. Large differences in the concentrations of the bovine immunoglobulin isotypes among the various pools of colostrum were positively correlated with concentration of these isotypes in the sera of the neonatal pigs fed these pools. Relative to their concentrations in colostrum, approximately 41% of the IgG1, 55% of the IgG2, 29% of the IgM, and 67% of the IgA was absorbed. The IgA was absorbed the best and IgM was least absorbed. Significant trends or differences in absorption were not observed among groups. Neonatal pigs given fresh colostrum, which had a higher fat content, had significantly more weight gain (P less than 0.05). This occurred, despite the fact that the fresh colostrum had the lowest concentration of bovine immunoglobulin. Serum half-lives for bovine IgG1 and IgG2 were significantly less than for porcine IgG (P less than 0.05), whereas the half-lives for bovine and porcine IgM and IgA were similar. De novo-synthesized immunoglobulins were detectable in serum after 6 days; IgM concentrations reached a maximum at 15 days in neonatal pigs given stored, but not fresh, colostrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal-neonatal immunoregulation in swine. II. Influence of multiparity on de novo immunoglobulin synthesis by piglets.

In the first-litter sows lower serum levels were found for all three Ig classes as compared to multiparous sows. The same was true for IgA in lacteal secretions and in piglet serum during the first days of life, while no differences were found for IgG levels. In contrast to these findings, IgM levels were found to be higher in lacteal secretions of first-litter sows and in piglet serum during the first days of life as compared to their counterparts. From three weeks after birth Igs found in piglet serum mainly originate from de novo synthesis. In this period piglets of first litter sows showed a higher IgA level up to the 6th week of life and higher IgG and IgM levels up to the end of the investigation period. Results are discussed in terms of maternal-neonatal immune regulation, focussing on the apparent suppressive role of maternally-derived IgG on total de novo Ig synthesis by suckling piglets.

Changes in the concentrations of serum IgG, IgA and IgM of sows throughout the reproductive cycle.

The concentrations of IgG, IgM and IgA in sera collected from 3855 sows (3208 pregnant and 647 lactating) at a single time point were determined. This experimental design allowed changes in serum immunoglobulin over the reproductive cycle to be studied without bias from seasonal influence. The concentrations of the three immunoglobulins changed independently during the reproductive cycle. Serum levels of IgM and IgG began a progressive postpartal decline during the 14th-17th week of gestation. At the onset of lactation serum IgG levels progressively increased while IgM levels continued to decline, the latter reaching their lowest level during the third week of lactation. In contrast to IgM and IgG, serum IgA levels increased 35% during weeks 14-17 of gestation and continued to increase throughout lactation, reaching their highest serum levels in the third week of lactation; the serum IgA concentration at this time was twice that observed during the first 13 weeks of gestation. Results of these studies allowed the reproductive cycle to be classified into four phases on the basis of serum immunoglobulin concentrations: (1) weeks 1-4 of gestation; (2) weeks 5-13 of gestation; (3) weeks 14-17 of gestation and (4) lactation.

The influence of age and breed on the concentrations of serum IgG, IgA and IgM in sows throughout the reproductive cycle.

The influence of age and breed on the concentrations of IgG, IgA and IgM in the sera of sows throughout the reproductive cycle was investigated in 4137 sows which had had 0-20 gestations and representing three breeds: Swedish Landrace, German Landrace and L-12 (= Swedish Landrace x Large White). Data revealed an increase in total immunoglobulins (Ig), IgM and IgG serum levels with increasing gestation number; the latter contributed greater than 80% to total Ig levels. IgG was significantly increased up to the fourth gestation, whereas IgM showed a significant increase only to the third gestation. IgA showed only minor differences. Age-dependent increases in serum IgM were ascribed to the increased probability of antigenic exposure during suckling, while failure to observe this change in serum IgA was ascribed to its role as a local Ig. Breed differences were observed to be significant for all three Ig's. It is concluded that establishment of group norms for serum Ig's should consider age and breed difference as well as stage of gestation or lactation.