On the emerging role of rabbit as human disease model and the instrumental role of novel transgenic tools.

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for human diseases, because of its size, which permits non-lethal monitoring of physiological changes and similar disease characteristics. Novel transgenic tools such as, the zinc finger nuclease method and the sleeping beauty transposon mediated or BAC transgenesis were recently adapted to the laboratory rabbit and opened new opportunities in precise tissue and developmental stage specific gene expression/silencing, coupled with increased transgenic efficiencies. Many facets of human development and diseases cannot be investigated in rodents. This is especially true for early prenatal development, its long-lasting effects on health and complex disorders, and some economically important diseases such as atherosclerosis or cardiovascular diseases. The first transgenic rabbits models of arrhythmogenesis mimic human cardiac diseases much better than transgenic mice and hereby underline the importance of non-mouse models. Another emerging field is epigenetic reprogramming and pathogenic mechanisms in diabetic pregnancy, where rabbit models are indispensable. Beyond that rabbit is used for decades as major source of polyclonal antibodies and recently in monoclonal antibody production. Alteration of its genome to increase the efficiency and value of the antibodies by humanization of the immunoglobulin genes, or by increasing the expression of a special receptor (Fc receptor) that augments humoral immune response is a current demand.

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.

The heterogeneity of bovine IgG2--VIII. The complete cDNA sequence of bovine IgG2a (A2) and an IgG1.

The complete cDNA sequences of two bovine IgGs were obtained by RT-PCR cloning. The first-strand cDNA was prepared from an animal homozygous (A2/A2) for IgG2a; the resulting sequences of the two bovine IgGs reported here were identified as IgG2a(A2) and IgG1. These sequences, and their deduced amino acid sequences, are compared to the previously reported partial protein sequences of IgG2a(A2) and IgG2(A1), two genomic DNA sequences of IgG2a and one genomic DNA sequence of IgG1. Data show that the two IgG2a allotypes (A1 and A2) differ in four regions: (a) region I-the site of the L-H bond in CH1; (b) region II-the middle hinge; (c) region III-a seven amino acid region at the beginning of the intradomain loop in CH3; and (d) region IV-an Arg-to-Glu exchange at the end of the same intradomain loop. The A1 allotype, which so remarkably distinguishes these allotypic variants, must result from differences in regions III and IV. The IgG1 sequence differs in the hinge region from the sequence reported previously and may represent an allotypic variant. We found no evidence to support the hypothesis that similarities between the CH3 domains of IgG2a(A2) and IgG1 result from gene conversion in the C-region of the bovine heavy chain locus.

The heterogeneity of bovine IgG2. VII. The phenotypic distribution of the A1 and A2 allotypes of IgG2a among beef cows with known clinical history.

A1 and A2 are allotypes of bovine IgG2a which differ significantly in their primary structure, allotope expression and the products of pepsin digestion. An analysis of 754 beef cows from 14 different breeds at the Meat Animal Research Center (MARC), Clay Center, NE, demonstrated a significant difference in the distribution of A1 and A2 among breeds but failed to find any correlation between the clinical disease history of the animals tested and their A-allotype. The proportion of all animals with either a history of infectious or respiratory disease (43.3 +/- 3.5 and 17 +/- 0, respectively) was the same among A1/A1, A1/A2 and A2/A2 animals. Similarly, there was no preferential association between allotype and clinical disease within any one breed. A very high incidence of A1 homozygotes was found among Angus (84%), Brown Swiss (100%), Limousin (87%), MARC I (87%) and Pinzgauers (88%). In contrast, Herefords had a high incidence of A2/A2 homozygotes (41%) as did Brahmans (46%) and Gelbveih (34%). The distribution of A1/A1, A1/A2 and A2/A2 animals within any breed was totally consistent with the concept that A1 and A2 represent Mendelian co-dominant alleles. These data suggest that, among vaccinated female beef cattle in a normal environment, A-allotypy plays no role in the propensity for clinical disease as defined in this study. It does not rule out such an association in non-vaccinated, severely stressed animals and in calves exposed to severe outbreaks of an infectious agent.

Functional restoration of the bursa of Fabricius following in ovo infectious bursal disease vaccination.

The primary role of the avian bursa of Fabricius is to provide an essential microenvironment for B-lymphocytes to diversify their immunoglobulin genes by gene hyperconversion. Infectious bursal disease (IBD) vaccination using intermediate plus vaccine strains can temporarily deplete the bursal follicles and interrupt the normal B-cell development, which is generally followed by B-cell repopulation and histological regeneration. To find evidence that functional restoration of the bursa of Fabricius occurs in addition to the histological regeneration, we have analysed the chB1 gene expression, which indicates active bursal B-lymphocytes, and also the surface expression of a carbohydrate structure Lewis(x), a marker which identifies those bursal B-lymphocytes that are undergoing gene hyperconversion. In ovo vaccination with an immune complex vaccine (IBDV-BDA) caused transient bursal destruction in both the SPF and the maternally protected broiler groups with differences evident in the starting time, the severity and the duration of the effect. After the depletion phase, signs of histological regeneration appeared together with chB1- and Lewis(x) expression indicating that B-lymphocytes were functionally active and the bursa of Fabricius was serving again as an efficient primary lymphoid organ providing an appropriate microenvironment for B-cell development.

The VH and CH immunoglobulin genes of swine: implications for repertoire development.

Swine have the largest number of IgG subclass genes of all species so far studied but have a single gene for IgA which occurs in two allelic forms that differ in hinge length. Swine also have constant region genes for C mu and C epsilon, but lack a gene homologous to that which encodes IgD in rodents and primates, despite the otherwise high degree of sequence similarity of all other swine CH genes with those of humans. Swine have < 20 VH genes, a single JH and perhaps a limited number of DH segments. Newborn piglets show preferential VH and DH usage and may use gene conversion as a mechanism for expanding their antibody repertoire. Despite the close similarity of their Ig gene sequences to humans, swine belong to the group of animals that includes rabbits, chickens and cattle when classified on the basis of B cell development. This group, unlike rodents and humans, have a single VH family, use hindgut follicles early in life (rather than bone marrow throughout life) to diversify their antibody repertoire and probably all use gene conversion. It is proposed that IgD may serve a function in repertoire development in rodents and humans which is unnecessary in the chicken-lagomorph-artiodactyl group. The diversity of immunoglobulins and immunoglobulin genes among species justifies the quest of veterinary immunologists to define the system for their species of interest rather than making extrapolations from mouse and human immune systems.

Bovine IgG2a antibodies to Haemophilus somnus and allotype expression.

Bovine IgG2a has been implicated in protection against pyogenic infections, including those caused by Haemophilus somnus. To further investigate the role of IgG2a in defense against H. somnus, IgG1 and IgG2a antibodies were purified from antiserum against an immunodominant 40 kDa outer membrane protein (p40) of H. somnus, which was previously shown to passively protect calves against H. somnus pneumonia. The passive protective capacity of anti-p40 IgG1 or IgG2a was evaluated in vivo in calves. Purified anti-p40 IgG1 or IgG2a was incubated with H. somnus for 15 min before intrabronchial inoculation of calves. Bacteria incubated with anti-p40 IgG1 or IgG2a were inoculated into one caudal lung lobe and bacteria incubated with IgG1 or IgG2a from the respective preimmunization serum were inoculated into the contralateral lobe. The volumes of pneumonia in the right and left lungs were determined 24 h later. The difference in volume of pneumonia with H. somnus preincubated in IgG1 pre- and postimmunization anti p40 was less (16 cm3, P = 0.298) than the difference in volume of pneumonia with H. somnus preincubated in IgG2a pre- and postimmunization anti p40 (30 cm3, P = 0.146). Although the differences in lesion size between pre- and postimmunization serum were not statistically significant, the trend suggests IgG2a may be more protective than IgG1. To examine this further, the peptide specificity of these IgG1 and IgG2a antibodies to p40 was examined. After limited proteolysis of p40, IgG2a antibodies reacted with 2 peptides not recognized by IgG1 antibodies. Other peptides were recognized by both isotypes. Since these studies suggested that IgG2a may be important in protection against infection, we then investigated some aspects of the role of the 2 IgG2a allotypes, A1 and A2. In retrospective studies of age differences in expression of IgG2a allotypes, no heterozygotes were detected in calves of 60 d old or less, and fewer heterozygotes were detected in calves 61-120 d old than in cattle older than 270 d (P < 0.01). In a subsequent prospective study of the time course of allotype expression, Holstein calves shown to be heterozygotes expressed the IgG2aA1 allotype early but the IgG2aA2 allotype was not usually detected until 3 to 4 mo of age. Thus, both the retrospective and the prospective studies showed age related differences in expression of the IgG2aA1 and A2 allotypes. This could have implication in protection.

Detection of Lawsonia intracellularis in Hungarian swine herds by polymerase chain reaction.

Pooled faecal samples and/or intestinal contents from pig carcasses were collected from 11 Hungarian swine farms and subjected to DNA extraction/ purification and subsequent polymerase chain reaction (PCR) in order to detect Lawsonia intracellularis, the aetiological agent of the porcine proliferative enteropathy complex. Specific PCR positivity was detected in 6 individual and 22 pooled samples out of 46, originating from eight herds. The PCR products of collected faecal samples hybridised in Southern blot hybridisation with the DNA of the type strain L. intracellularis NCTC 12657. This is the first confirmed detection of L. intracellularis by PCR in Hungary.

Immunoglobulins, antibody repertoire and B cell development.

Swine share with most placental mammals the same five antibody isotypes and same two light chain types. Loci encoding lambda, kappa and Ig heavy chains appear to be organized as they are in other mammals. Swine differ from rodents and primates, but are similar to rabbits in using a single VH family (VH3) to encode their variable heavy chain domain, but not the family used by cattle, another artiodactyl. Distinct from other hoofed mammals and rodents, Ckappa:Clambda usage resembles the 1:1 ratio seen in primates. Since IgG subclasses diversified after speciation, same name subclass homologs do not exist among swine and other mammals unless very closely related. Swine possess six putative IgG subclasses that appear to have diversified by gene duplication and exon shuffle while retaining motifs that can bind to FcgammaRs, FcRn, C1q, protein A and protein G. The epithelial chorial placenta of swine and the precosial nature of their offspring have made piglets excellent models for studies on fetal antibody repertoire development and on the postnatal role of gut colonization, maternal colostrum and neonatal infection on the development of adaptive immunity during the "critical window" of immunological development. This chapter traces the study of the humoral immune system of this species through its various eras of discovery and compiles the results in tables and figures that should be a useful reference for educators and investigators.

Five putative subclasses of swine IgG identified from the cDNA sequences of a single animal.

We report the sequences of more than 40 partial and complete swine C gamma cDNAs obtained by PCR cloning of first strand cDNA, and from a cDNA expression library, all from a single animal. These seem to represent five IgG subclasses, that can be grouped into two clusters; one contains IgG1 and IgG3 and the other, IgG2a, IgG2b and IgG4. IgG2a and IgG2b differ by only three amino acids, but single strand conformational polymorphism analyses of PCR-amplified IgG2-specific segments in animals of different breeds, argues for their putative subclass status. Major subclass differences are found in the hinge and C gamma 3, but differences in upper hinge length, associated with segmental flexibility in the IgGs of other species, are absent. All subclasses have identical middle hinge segments that can accommodate three interheavy chain disulfide bridges. The putative swine IgG subclasses have their greatest similarity with those of the human except for the near absence of hinge region variation. Swine subclasses such as ruminant IgG2a, have a lower hinge deletion which in human IgG1, contains one of the motifs believed necessary for interaction with Fc gamma Rs. IgG1 was the most frequently encountered subclass cDNA (25 of 43) and the single swine-mouse hybridoma tested had a sequence identical to IgG1. Partial sequence analyses of genomic clones identified one clone identical with the IgG1/IgG3 subclass cluster, two identical to the IgG2b/IgG4 subclass group, and two identical to each other but different from any of the expressed sequences reported here. Genomic blots suggest that up to eight C gamma genes are present in the genome.

Expressed swine VH genes belong to a small VH gene family homologous to human VHIII.

The sequences of 34 swine H chain V-regions expressed with either IgG, IgA, or IgM C regions in adult swine and newborn piglets are described and compared. Sixteen of these V-regions were cloned by using a specific leader primer whereas 18 were cloned by anchored PCR. According to the operational criterion of VH family classification, i.e., > 80% DNA sequence similarity, all sequences belong to a homogeneous VH gene family. A total of 31 additional VH-bearing C mu clones obtained by anchored PCR from colostrum-deprived newborn piglets hybridized equally with a probe for C mu and a pan-VH probe. When the consensus sequence of the expressed swine VH genes were compared with those of VH gene for humans (VHI, II, III), mouse (VHI, II, III), rabbit, and chicken, swine VH genes seem to have common ancestry with the human VHIII family, rabbit VH genes, and the single functional VH gene of the chicken. The leader peptides of all clones obtained by anchored PCR showed < 1% variability, and the deduced amino acid sequences from aa4 to aa25 in all 34 clones are identical. Framework (FR)1 and FR2 are conserved whereas FR3 shows greater variability. A total of 23 of 30 JH sequences were identical, suggesting preferential use of one JH. This frequent, putative JH sequence is not similar to any JH gene in humans. Identical genomic Southern hybridization patterns, each with 13 bands of differing intensity and regardless of stringency, were obtained when either a leader or a pan-specific VH probe was used. Analyses of individual genomic bands by single strand conformational polymorphism and sequence analysis suggest that the number of VHIII-related genes in the swine genome is < 20.

Cloning and characterization of the bovine MHC class I-like Fc receptor.

In the cow, maternal immunity is exclusively mediated by colostral Igs, but the receptor responsible for the IgG transport has not yet been identified. The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. We now report the cloning and characterization of the bovine FcRn (bFcRn). The cDNA and deduced amino acid sequences show high similarity to the FcRn in other species, and it consists of three extracellular domains, a hydrophobic transmembrane region, and a cytoplasmic tail. Despite the high similarity of the extracellular domains with other species, the bovine cytoplasmic tail is the shortest thus far analyzed. Aligning the known FcRn sequences, we noted that the bovine protein shows a 3-aa deletion compared to the rat and mouse sequences in the alpha1 loop. Furthermore, we found a shorter transcript of the bFcRn reflecting an exon 6-deleted mRNA, which results from an inadequate splice acceptor site in intron 5 and produces a transmembrane-deficient molecule, as was previously demonstrated in the related MHC class I gene family in mouse and humans. The presence of bFcRn transcripts in multiple tissues, including the mammary gland, suggests their involvement both in IgG catabolism and transcytosis.

The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C alpha intron.

Recently published genomic and cDNA sequences for porcine IgA suggested that the splice acceptor site in the C alpha 1-C alpha 2 intron was an AA rather than an AG dinucleotide. This possibility was tested in an in vitro HeLa cell splicing system using an RNA substrate corresponding to the genomic DNA with the putative AA splice site. Data indicated that splicing occurred at a cryptic AG site 12 nucleotides into the C alpha 2 domain rather than at the AA site. The possibility that swine B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the segment from the first C alpha 1 nucleotide to nucleotide 532 in C alpha 2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequencing gel revealed two polynucleotide products in most samples that differed by the expected 12 nucleotides, suggesting that swine could use both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparent upstream AA site. However, when the genomic DNA from an animal expressing only the longer polynucleotide was cloned and sequenced, the upstream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgAa and IgAb, which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgAb) variant, in which the downstream cryptic splice site is used, as well as a "normal-hinged" (six amino acids, IgAa) variant. Evidence that IgAa and IgAb are allelic was confirmed by genotypic analyses of progeny from matings of IgAa/IgAb heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA levels were similar in animals homozygous for each variant.