Establishment of a resource population of SLA haplotype-defined Korean native pigs

The highly polymorphic porcine major histocompatibility complex (MHC), or the swine leukocyte antigens (SLA), has been repeatedly associated with variations in swine immune response to pathogens and vaccines as well as with production traits. The SLA antigens are also important targets for immunological recognition of foreign tissue grafts. We recently established a resource population of Korean native pigs as models for human transplantation and xenotransplantation research. In this study, 115 animals derived from three generations of the Korean native pigs were genotyped for three SLA class I (SLA-2, SLA-3 and SLA-1) and three SLA class II loci (DRB1, DQB1, DQA) using PCR with sequence-specific primers (PCR-SSP) at the allele group resolution. A total of seven SLA haplotypes (Lr-5.34, Lr-7.23, Lr-31.13, Lr-56.23, Lr-56.30, Lr-59.1, Lr-65.34), comprising six unique class I and five unique class II haplotypes, were characterized in the founding animals. Class I haplotype Lr-65.0 and class II haplotype Lr-0.34 were novel; and together with Lr-56.0 these haplotypes appeared to be breed-specific. In the progeny population, Lr-7.23 and Lr-56.30 appeared to be the most prevalent haplotypes with frequencies of 34.7% and 31.6%, respectively; the overall homozygosity was 27.4%. This resource population of SLA-defined Korean native pigs will be useful as large animal models for various transplantation and xenotransplantation experiments, as well as for dissecting the roles of SLA proteins in swine disease resistance and production traits.     

Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations

The highly polymorphic swine leucocyte antigen ( SLA ) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci ( SLA-1 , SLA-3 and SLA-2 ) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations ( n =  202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 ( SLA-1 *01XX- SLA-3 *01XX- SLA-2 *01XX) and Lr-4.0 ( SLA-1 *04XX- SLA-3 *04XX- SLA-2 *04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined ( n  =   162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines.     

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.     

Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes

The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.     

Microsatellite diversity and crossover regions within homozygous and heterozygous SLA haplotypes of different pig breeds

Our aim was to investigate microsatellite (MS) diversity and find crossover regions at 42 polymorphic MS loci in the swine leukocyte antigen (SLA) genomic region of 72 pigs with different well-defined homozygous and heterozygous SLA haplotypes. We analyzed the genetic polymorphisms of 42 MS markers in 23 SLA homozygous-heterozygous, common pig breeds with 12 SLA serological haplotypes and 49 National Institutes of Health (NIH) and Clawn homozygous-heterozygous miniature pigs with nine SLA serological or genotyped haplotypes including four recombinant haplotypes. In comparing the same and different haplotypes, both haplospecific patterns and allelic variations were observed at the MS loci. Some of the shared haplotype blocks extended over 2 Mb suggesting the existence of strong linkage disequilibrium (LD) in the entire SLA region. Crossover regions were easily defined by the MS markers within the class I and/or III region in the NIH and Clawn recombinant haplotypes. The present haplotype comparison shows that our set of MS markers provides a fast and cost-efficient alternative, or complementary, method to the serological or sequence-based determination of the SLA alleles for the characterization of SLA haplotypes and/or the crossover regions between different haplotypes.     

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line,Microminipig

Microminipigs are extremely small‐sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo‐ and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA‐defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA‐1, SLA‐2 and SLA‐3) and two class II (SLA‐DRB1 and SLA‐DQB1) genes of 14 parental Microminipigs using a high‐resolution nucleotide sequence‐based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp‐31.0 (SLA‐1*1502–SLA‐3*070102–SLA‐2*1601) and Hp‐0.37 (SLA‐DRB1*0701–SLA‐DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.     

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F ₂ descendants of F ₁ Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA‐1 , SLA‐2 , SLA‐3 ) and class II ( SLA‐DRB1 , SLA‐DQB1 , SLA‐DQA ) genes of 27 purebred Pietrain pigs using a combination of the high‐resolution sequence‐based typing (SBT) method and a low‐resolution (Lr) PCR‐based method using allele‐group, sequence‐specific primers (PCR‐SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr‐43.0 ( SLA‐1 *11XX– SLA‐3 *04XX– SLA‐2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr‐0.14 [ SLA‐DRB1 *0901– SLA‐DQB1 *0801– SLA‐DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr‐Pie‐0.1 [ SLA‐DRB1 *01XX/be01/ha04– SLA‐DQB1 *05XX– SLA ‐ DQA*blank] and Lr‐Pie‐0.2 [ SLA‐DRB1 *06XX– SLA‐DQB1 *03XX– SLA‐DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR‐SSP‐based assays represents a rapid and cost‐effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease‐resistant pigs in the context of SLA antigens to improve overall swine health.     

Bioenergetics of camel crickets (Ceuthophilus carlsbadensis,C. longipes and C. Conicaudus) from carlsbad caverns national park,new mexico

• 1.1. Camel crickets in Carlsbad Caverns National Park exhibit linear long-term weight loss patterns for combined sexes of 1.05 mg/hr for Ceuthophilus carlsbadensis, 0.261 mg/hr for C. conicaudus, and 0.321 mg/hr for C. longipes.
• 2.2. From these patterns, maximal foraging intervals for females and males, respectively, of 5.1 and 4.4 days for C. carlsbadensis, 4.6 and 5.7 days for C. longipes, and 5.0 and 4.2 days for C. conicaudus were predicted.
• 3.3. Calculated metabolic rates (cal/hr) of 1.04 for C. carlsbadensis and 0.52 for C. longipes were half that predicted for epigean species of similar size.

     

Carcass proximate composition of juvenile white sturgeon (acipenser transmontanus)

• 1.1. Body wt, carcass wt, liver wt, gonad wt, total length, and carcass proximate compositions of 8- to 32-month-old cultured sturgeon were significantly affected (P < 0.05) by ages.
• 2.2. Correlation coefficients were significant between age and body wt (r = 0.96), carcass wt (r = 0.96), total length (r = 0.92), liver wt (r = 0.88), gonad wt (r = 0.76), gonadosomatic index (r = 0.52), carcass moisture (r = −0.93), crude protein (r = 0.62) and lipid (r = 0.57).
• 3.3. The carcass proximate compositions of cultured sturgeon were 72.1–79.4% moisture, 13.5–17.5% crude protein, 3.4–7.4% crude lipid, and 4.2–5.0% ash.
• 4.4. A high variability in the body measurements and carcass proximate compositions of some cultured sturgeon suggested high potential for genetic and nutrition improvements in culturing these fish in hatcheries.

     

Spatial arrangement of pig MHC class I sequences

Bacterial artificial chromosome (BAC) clones were assigned within the pig major histocompatibility complex (Mhc) by polymerase chain reaction-screening and Southern blot hybridization using sequence-tagged site (STS) markers and BAC end-rescued sequences. In all, 35 BAC clones were discovered containing 12 anchor genes of the SLA class I region and two genes of the SLA class III region. Twenty of these 35 clones comprised two distinct class I gene clusters, each spanning about 100 kilobases. One cluster enclosed three class I related genes (SLA-6 to -8) and two genes (MIC-1 and MIC-2) more distantly related to class I. The other cluster enclosed typical class I genes, of which three (SLA-1, -2, and -3) were transcribed by fibroblasts homozygous for the H01 haplotype which we used to construct a pig BAC library. Ordered clones are certainly helpful in isolating agronomically, biologically, and medically important genes. They would also be useful for inducing genetic modifications in pig cell lines.     

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype ({"type":"entrez-nucleotide","attrs":{"text":"AF479533","term_id":"28194589","term_text":"AF479533"}}AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.     

Sequence of the pig major histocompatibility region containing the classical class I genes

A segment comprising 307,078 nucleotides of the pig major histocompatibility complex (SLA) was completely sequenced. The segment corresponded to the entire SLA classical class I-containing region of the serologically defined SLA H01 haplotype. In all, 11 genes were characterized, comprising 7 class I genes located on the centromeric part of the sequence (SLA-1, 2, 3, 4, 5, 9, and 11) and 4 ring finger-related family genes located on its telomeric part. No member of one family was intermingled with a member of the other or with any third-party gene. All class I genes except SLA-11 were similarly orientated. The SLA-1, 2, and 3 genes displayed both promoter and overall coding regions compatible with normal functions. The SLA-4, 11, and 9 genes were considered pseudogenes because they exhibited marked anomalies. Although the SLA-5 gene had a complete coding region, it displayed mutations in promoter elements which could modify its expression. The great molecular similarity observed among the class I genes extended far outside them, and resulted from segmental duplications. The ring finger genes exhibited great homology with their human counterparts. In pig, one of these genes appeared to correspond to a complete gene which in humans is probably a pseudogene. In all, the 11 genes characterized span about 20% of the total sequence. The remaining 80% consists of interspersed repeat elements. The present results, together with the sequence previously reported involving the SLA class I-related genes, open the way for a better understanding of pig MHC organization.     

Molecular characteristics of the <Emphasis Type="Italic">SLA-2</Emphasis> gene from Chinese Hebao pigs

To study the molecular characteristics of swine leukocyte antigen (SLA) class I from the Hebao pig, a rare inbreed in China, a pair of primers was designed to amplify the SLA-2 gene (SLA-2-HB) and then the molecular characteristics of the gene were analyzed by computer. After cloning, sequencing and computer analysis, four SLA-2-HB alleles were found, all of 1119 bp. Sites 3–1097 were an open reading frame encoding 364 amino acids with two sets of intra-chain disulfide bonds comprising four cysteines situated in sites 125, 188, 227 and 283. By alignment of SLA-2-HB sequences with other SLA-2 alleles in the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank database, nine key variable amino acid sites were found in the extracellular domain of the SLA-2-HB alleles at sites 23(F), 24(I), 43(A), 44(K), 50(Q), 73(N), 95(I), 114(R) and 216(S), which could be used to differentiate other SLA-2 alleles. The amino acid identities between SLA-2-HB and other SLA-2, SLA-3 and SLA-1 alleles were 87.1–97.0%, 85.0–93.9% and 83.3–88.6%, respectively. The phylogenetic tree of SLA-2-HB showed that it was relatively independent of the other SLA-2 genes. Furthermore, the SLA-2-HB alleles were similar to HLA-B15 and HLA-A2 functional domains and preserved some functional sites of HLA-A2. It was concluded that SLA-2-HB is an allele of SLA-2 and that the Hebao pig might have evolved independently in China.     

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Levels of aflatoxin B1, bacteria and fungi in feed and food in 1971 — 1987

Totally 39% out of 8371 feed and their component samples were contaminated by aflatoxin B₁. Mean contamination was 36μg/kg with maximum yield 10100 μg/kg. Contamination of samples by total count of organisms, mean contamination and maximum yield, respectively was: 1) bacteria 99%, 2.2×10⁶, 2.4×10⁸; 2) proteolytic bacteria 94%, 1.2×10⁵, 3.0×10⁶;3) moulds 98%, 1.3×10⁵, 9.0×10⁶; 4) yeasts 44 %, 3.3×10⁴, 3.6×10⁶. The samples were contaminated in 92 % byAspergillus spp, in 71% byAspergillus flavus, in 83% byPenicillium spp, and in 20% byFusarium spp with mean contamination 8.3×104, 1.1×10³, 4.2×10⁴, 5.0×10³ , and maximum yield 6.8×10⁶, 1.0×10⁵, 5.0×10⁶, 1.5×10⁶, respectively. Totally 8.5% of strains were aflatoxinogenic and 4.4% of the strains were isolated from feed and 21 % of the strains from grain/nut.     

Nucleotide sequence analysis of <Emphasis Type="Italic">S</Emphasis>-locus genes to unify <Emphasis Type="Italic">S</Emphasis> haplotype nomenclature in radish

Radish, belonging to the family Brassicaceae, has a self-incompatibility which is controlled by multiple alleles on the S locus. To employ the self-incompatibility in an F₁ breeding system, identification of S haplotypes is necessary. Since collection of S haplotypes and determination of nucleotide sequences of SLG, SRK, and SCR alleles in cultivated radish have been conducted by different groups independently, the same or similar sequences with different S haplotype names and different sequences with the same S haplotype names have been registered in public databases, resulting in confusion of S haplotype names for researchers and breeders. In the present study, we developed S homozygous lines from radish F₁ hybrid cultivars in Japan and determined the nucleotide sequences of SCR, the S domain and the kinase domain of SRK, and the SLG of a large number of S haplotypes. Comparing these sequences with our previously published sequences, the haplotypes were ordered into 23 different S haplotypes. The sequences of the 23 S haplotypes were compared with S haplotype sequences registered by different groups, and we suggested a unification of these S haplotypes. Furthermore, dot-blot hybridization using SRK allele-specific probes was examined for developing a standard method for S haplotype identification.     

Bovine serum sialic acid: Age-related changes in type and content

• 1.1. The sialic acid content of newborn calf serum (4.8 μmol/ml) is approx. 3-fold higher than that of mature animals (1.4 μmol/ml) and decreases to 2.4 μmol/ml at 20 days of age. Colostrum-fed and colostrum-deprived calves have similar levels of sialic acid from birth to 14 days of age.
• 2.2. The high level of sialic acid in newborn calf serum is due predominantly to N-acetylneuraminic acid, since this sialic acid accounts for 93% of the total and since <5% of the sialic acid is O-acetylated.
• 3.3. Comparison of day 0 and day 20 serum by gel filtration and by SDS polyacrylamide gel electrophoresis demonstrates that the increase in sialic acid is associated with increased production and/or sialylation of components with MW of 45–60 kDa.
• 4.4. A high percentage (64%) of the sialic acid in newborn calf serum is detected with the lipid-linked sialic acid assay, relative to 20 day old (25%) or mature (18%) animals.
• 5.5. This indicates that the glycoproteins of newborn calf serum are more efficiently extracted under the conditions of this assay than glycoproteins of mature serum.

     

Ex Vivo SIV-Specific CD8 T Cell Responses in Heterozygous Animals Are Primarily Directed against Peptides Presented by a Single MHC Haplotype

The presence of certain MHC class I alleles is correlated with remarkable control of HIV and SIV, indicating that specific CD8 T cell responses can effectively reduce viral replication. It remains unclear whether epitopic breadth is an important feature of this control. Previous studies have suggested that individuals heterozygous at the MHC class I loci survive longer and/or progress more slowly than those who are homozygous at these loci, perhaps due to increased breadth of the CD8 T cell response. We used Mauritian cynomolgus macaques with defined MHC haplotypes and viral inhibition assays to directly compare CD8 T cell efficacy in MHC-heterozygous and homozygous individuals. Surprisingly, we found that cells from heterozygotes suppress viral replication most effectively on target cells from animals homozygous for only one of two potential haplotypes. The same heterozygous effector cells did not effectively inhibit viral replication as effectively on the target cells homozygous for the other haplotype. These results indicate that the greater potential breadth of CD8 T cell responses present in heterozygous animals does not necessarily lead to greater antiviral efficacy and suggest that SIV-specific CD8 T cell responses in heterozygous animals have a skewed focus toward epitopes restricted by a single haplotype.     

The effect of cryopreservation and various cryodiluents on allozyme variation of glucose phosphate isomerase in the F1 progency of african catfish (clarias gariepinus)

• 1.1. African sharptooth catfish individuals, heterozygous for glucose phosphate isomerase (GPI-2), were selected as broodstock by using horizontal starch-gel electrophoresis.
• 2.2. Ova of one heterozygous female were inseminated with cryopreserved and fresh milt from a corresponding male.
• 3.3. Significant deviations from expected Hardy-Weinberg proportions occured for offspring obtained by using cryopreserved milt.
• 4.4. Differences in genotypic variation seems to relate to different cryodiluents and the fertility thereof.
• 5.5. Selection of breeding stock for aquacultural practices based on the above information is discussed.