Bovine Class Research Articles

Comparison of Antimicrobial Susceptibility of Escherichia Coli Isolated from Fecal Poultry and Bovine Housed in Tunisian Farms; Phylogroup Diversity and Detection of Tetracycline and Sulfonamides Resistant Genes With Integron Class1

Little detailed documentation researched the excessive use of antimicrobials such as tetracycline and sulfonamides in veterinary medicine in Tunisia and more studies are needed. A total of 58 of commensal Escherichia coli isolates recovered from fecal samples of healthy poultry (n=31) and bovin (n=27) recovered from farms in Tunisia were examinated for 20 antimicrobial as well as, researched the presence of integron, variable regions (VRs), phylogroupes, tetracycline (tetA, tetB et tetC) and sulfonamides(sul1, sul2, sul3) resistance genes. The most frequently resistance in poultry origin were to tetracycline (94.3%), sulfonamide (70.69%), nalidixic acid (61.29), amoxicillin (58%), to trimthetoprim-sulfamethoxazole and streptomycin with the same rate (64.51%), ticarcilline (58%). Whereas, the bovine isolates were most resistant to streptomycin (55.5%), to amoxicillin (18.5), to tetracycline (37%), and have a moderate same rates to kanamycine, to trimthetoprim-sulfamethoxazole 11.11, to nalidixic acid and to sulfonamide 7.4%, For poultry and bovine class 1 integron were detected in 20, 6 isolates, respectively as well as class 2 integron were found in 2 and 1 isolates, respectively. Class 1 integrons were significantly associated with poultry origin (p=0.001). For poultry sul1, sul2, and sul3 genes were detected in 14 (46.2 %), 7 (23.8 %), and 4 (8.9 %) resistant isolates, respectively. Whereas, for bovine 5 isolates were resistant to sulfonamide and sul1 and sul2 genes were detected in 4 and 1 isolates with absence of sul3 genes. and tetracycline genes tetA, tetB genes were observed in 27 (84.37 %) and 8 (25%) resistant isolates, respectively while, TetC was not detected amongst our isolates. Seven arrangement gene cassette were detected; dfrA1-satA1-aadA1 in one identical DNA fragments with approximate size of 2000 bp and six arrangements of resistance gene cassettes of class 1 integron were detected; dfrA1+aadA1 (5isolates); for dfrA17+aadA1, dfrA12+ orfF+aadA2 each one two 2isolates; one isolate for aadA1 and dfrA5, respectively. In poultry, 16 isolates were found to belong to phylogroup A (sub- groupA1: 12, sub- groupA0: 4); 9 to B1, 1 to B2 and 5 to phylogroup D. However, in bovine 9 isolates have the phylogroups A1, 7 isolates B1, 4 isolates B2, and 3 isolates found to phylogroup D. Our results showed that the prevalence of resistance in E. coli isolates from poultry was much higher than that in bovin.

Identification of a DRB3*1101-restricted CD4 T cell response against bovine respiratory syncytial virus

Abstract A better understanding of the immune response elicited by bovine respiratory syncytial virus (BRSV) vaccines is needed for vaccine improvement. Although killed-BRSV vaccines are available as part of multivalent products, their efficacy is controversial. We screened for BRSV-specific T cell responses in cows with a history of annual vaccinations. We defined the minimum BRSV F peptide 261–270 (INDMPITNDQ), which elicits IFN-γ from CD4 T cells in four of fourteen cows tested. The response is restricted by the bovine class II MHC molecule DRB3. All four cows share the DRB3*1101 allele. We are currently developing a specific DRB3*1101:INDMPITNDQ tetramer to facilitate further characterization of this response.

Fine-mapping host genetic variation underlying outcomes to Mycobacterium bovis infection in dairy cows

BackgroundSusceptibility to Mycobacterium bovis infection in cattle is governed in part by host genetics. However, cattle diagnosed as infected with M. bovis display varying signs of pathology. The variation in host response to infection could represent a continuum since time of exposure or distinct outcomes due to differing pathogen handling. The relationships between host genetics and variation in host response and pathological sequelae following M. bovis infection were explored by genotyping 1966 Holstein-Friesian dairy cows at 538,231 SNPs with three distinct phenotypes. These were: single intradermal cervical comparative tuberculin (SICCT) test positives with visible lesions (VLs), SICCT-positives with undetected visible lesions (NVLs) and matched controls SICCT-negative on multiple occasions.ResultsRegional heritability mapping identified three loci associated with the NVL phenotype on chromosomes 17, 22 and 23, distinct to the region on chromosome 13 associated with the VL phenotype. The region on chromosome 23 was at genome-wide significance and candidate genes overlapping the mapped window included members of the bovine leukocyte antigen class IIb region, a complex known for its role in immunity and disease resistance. Chromosome heritability analysis attributed variance to six and thirteen chromosomes for the VL and NVL phenotypes, respectively, and four of these chromosomes were found to explain a proportion of the phenotypic variation for both the VL and NVL phenotype. By grouping the M. bovis outcomes (VLs and NVLs) variance was attributed to nine chromosomes. When contrasting the two M. bovis infection outcomes (VLs vs NVLs) nine chromosomes were found to harbour heritable variation. Regardless of the case phenotype under investigation, chromosome heritability did not exceed 8% indicating that the genetic control of bTB resistance consists of variants of small to moderate effect situated across many chromosomes of the bovine genome.ConclusionsThese findings suggest the host genetics of M. bovis infection outcomes is governed by distinct and overlapping genetic variants. Thus, variation in the pathology of M. bovis infected cattle may be partly genetically determined and indicative of different host responses or pathogen handling. There may be at least three distinct outcomes following M. bovis exposure in dairy cattle: resistance to infection, infection resulting in pathology or no detectable pathology.

Haplotype distribution in the class I sirtuin genes and their associations with ultrasound carcass traits in Qinchuan cattle (Bos taurus)

Class I sirtuin genes including SIRT1, SIRT2 and SIRT3, are members of the nicotinamide adenine dinucleotide (NAD)-dependent family of histone deacetylases, and play essential roles in senescence, metabolism, and apoptosis. This study was conducted to detect potential polymorphisms of the bovine class I sirtuin genes and explore their relationships with ultrasound carcass traits in Qinchuan cattle. Four non-coding mutations in the 3′UTR (SIRT1: g.25751A > C, SIRT1: g.25846A > G, SIRT2: g.19676G > A and SIRT3: g. 25702C > T) and three mutations in exons (SIRT2: g.4062C > T; SIRT2: g.4406C > T and SIRT3: g.25557A > G) were identified in 468 individuals of Qinchuan cattle. Chi-square tests showed that g.25751A > C, g.19676G > A, and g.25702C > T were in Hardy-Weinberg disequilibrium (χ2 < χ0.052). The statistical analyses indicated that six SNPs were significantly associated with the ultrasound carcass traits (P < 0.05) except g.4062C > T (SIRT2) (P > 0.05). These results indicate that the variations in the class I sirtuin genes and their corresponding genotypes may be considered as molecular markers for economic traits in cattle breeding.

Vitamin B12 and homocysteine levels in blood of dairy cows during subacute ruminal acidosis

Abstract. The aim of this study was to investigate the variations of vitamin B12 and homocysteine in blood of dairy cows during subacute ruminal acidosis (SARA). On 228 subjects ruminal liquid was collected through rumenocentesis technique and rumen pH was immediately measured by a portable pH-meter. On the basis of pH values all cows were classified (bovine class) in Group A (animals with rumen pH&gt;5.7), Group B (animals with rumen pH between 5.6 and 5.7) and Group C (animals with rumen pH&lt;5.6). In relation to the acidosis risk depending on the rumen pH (herd class), the herds were classified in Group 1 (normal herds: less than 33 % cows with rumen pH&lt;5.8), Group 2 (critical herds: more than 33 % cows with rumen pH between 5.5 and 5.8) and Group 3 (acidosis herds: more than 33 % cows with rumen pH&lt;5.5). On blood samples, collected by jugular venipuncture, vitamin B12 and homocysteine were measured by chemiluminescent immunological tests. One-way analysis of variance (ANOVA), followed by Bonferroni test, showed significant differences (P&lt;0.05) for vitamin B12 in bovine class and significant differences (P&lt;0.05) for homocysteine in herd class. The influence of rumen pH values resulted in adequate vitamin B12 and homocysteine levels to meet microbial and cow requirements and fatty acids modifications in dairy cows affected by SARA. Moreover, the increase of vitamin B12 could be due to the presence of analogues which interfere with the transport of the vitamin. These findings provide more information on blood modifications during SARA.

Determination of trace elements in bovine semen samples by inductively coupled plasma mass spectrometry and data mining techniques for identification of bovine class

The reproductive performance of cattle may be influenced by several factors, but mineral imbalances are crucial in terms of direct effects on reproduction. Several studies have shown that elements such as calcium, copper, iron, magnesium, selenium, and zinc are essential for reproduction and can prevent oxidative stress. However, toxic elements such as lead, nickel, and arsenic can have adverse effects on reproduction. In this paper, we applied a simple and fast method of multi-element analysis to bovine semen samples from Zebu and European classes used in reproduction programs and artificial insemination. Samples were analyzed by inductively coupled plasma spectrometry (ICP-MS) using aqueous medium calibration and the samples were diluted in a proportion of 1:50 in a solution containing 0.01% (vol/vol) Triton X-100 and 0.5% (vol/vol) nitric acid. Rhodium, iridium, and yttrium were used as the internal standards for ICP-MS analysis. To develop a reliable method of tracing the class of bovine semen, we used data mining techniques that make it possible to classify unknown samples after checking the differentiation of known-class samples. Based on the determination of 15 elements in 41 samples of bovine semen, 3 machine-learning tools for classification were applied to determine cattle class. Our results demonstrate the potential of support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF) chemometric tools to identify cattle class. Moreover, the selection tools made it possible to reduce the number of chemical elements needed from 15 to just 8.

Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin

The comparative pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were investigated in lactating cows and beef steers. The plasma elimination half-life of either enrofloxacin or ciprofloxacin was shorter in cows than in steers. The overall production of ciprofloxacin was slightly higher in steers than in cows (metabolite ratio: 64% and 59%, respectively). There was no significant difference in plasma protein binding of enrofloxacin between cows (percent bound: 59.4%) and steers (percent bound: 60.8%). Ciprofloxacin was more extensively bound to plasma proteins in steers (percent bound: 49.6%) than in cows (percent bound: 33.8%). The steady state volume of distribution of enrofloxacin is comparable in cows (1.55 L/kg) and steers (1.59 L/kg). Within either bovine class, plasma elimination half-life of enrofloxacin and ciprofloxacin are comparable, while plasma protein binding was higher for enrofloxacin than for ciprofloxacin. Ciprofloxacin was more concentrated in milk than enrofloxacin.

Comparison of bovine lymphocyte antigen DRB3.2 allele frequencies between two subpopulations of Iranian holstein cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune responses by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. Since different alleles favor the binding of different peptides, DRB3 has been extensively evaluated as a candidate marker for associations with various bovine diseases and immunological traits. Therefore, in this study, the genetic diversity of the bovine class II DRB3 locus in the two Iranian subpopulations of Holstein cattle (Moghan farm n = 250 and Razavi farm n = 175) was investigated by polymerase chain reactionrestriction fragment length polymorphism method (PCR-RFLP). Bovine DNA was isolated from whole blood. A hemi-nested PCR followed by digestion with restriction endonucleases RsaI, HaeIII and BstYI was conducted on the DNA. The results indicated that exon 2 of the BoLA-DRB3 gene is highly polymorphic in the two populations, and the frequency of BoLA-DRB3 depends on breed. On the other hand the presence of BoLA-DRB3*8, *24, and *16 alleles with high frequency in BoLA-DRB3.2 system, can be used as appellative index for nominate Holstein.

YAC/BAC contig spanning the MHC class III region of cattle

A contig of the class III region of the bovine major histocompatibility complex (MHC) was established from bacterial and yeast artificial chromosomes using PCR and BAC-end sequencing. The marker content of individual clones was determined by gene and BAC-end specific PCR, and the location of genes and BAC-ends was confirmed analyzing somatic hybrid cells. A comparative analysis indicated that the content and order of MHC class III genes is strongly conserved between cattle and other mammalian species. Fluorescence in situ hybridization localized the bovine class III region to BTA23q21→q22. The results show that the collection of sequenced BAC-ends is a powerful resource for generating high-resolution comparative chromosome maps.

Identification of Bovine Lymphocyte Antigen DRB3.2 Alleles in Iranian Golpayegani Cattle by DNA Test

The bovine lymphocyte antigen (BoLA)-DRB3 gene encodes cell surface glycoproteins that initiate immune responses by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. Since different alleles favour the binding of different peptides, DRB3 has been extensively evaluated as a candidate marker for associations with various bovine diseases and immunological traits. For that reason, the genetic diversity of the bovine class II DRB3 locus was investigated by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). This study describes genetic variability in the BoLA-DRB3 in Iranian Golpayegani Cattle. Iranian Golpayegani Cows (n = 50) were genotyped for bovine lymphocyte antigen (BoLA)-DRB3.2 allele by polymerase chain reaction and restriction fragment length polymorphism method. Bovine DNA was isolated from aliquots of whole blood. A two-step polymerase chain reaction followed by digestion with restriction endonucleases RsaI, HaeIII and BstYI was conducted on the DNA from Iranian Golpayegani Cattle. In the Iranian Golpayegani herd studied, we identified 19 alleles.DRB3.2×16 had the highest allelic frequency (14%), followed by DRB3.2×7 (11%). Six alleles (DRB3.2×25, ×24, ×22, ×20, ×15, ×3) had frequencies = 2%. Although additional studies are required to confirm the present findings, our results indicate that exon 2 of the BoLA-DRB3 gene is highly polymorphic in Iranian Golpayegani Cattle. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 12 : 1691-1695)

Cloning and characterization of the bovine class 1 and class 2 insulin-like growth factor-I mRNAs

Insulin-like growth factor-I (IGF-I) is an important regulator of growth, development, and metabolism, and is the primary mediator of the growth-promoting activity of growth hormone (GH) in animals. In several species, the IGF-I polypeptide is generated from IGF-I mRNA containing either exon 1 (class 1 IGF-I mRNA) or exon 2 (class 2 IGF-I mRNA) as the leader exon. The objectives of this study were to identify class 1 and class 2 IGF-I mRNAs in cattle and to compare their expression in different tissues, their response to GH, and their translational efficiency. Three class 1 IGF-I cDNAs corresponding to three different transcription start sites in exon 1 and one class 2 IGF-I cDNA were identified from adult cattle liver using 5′-rapid amplification of cDNA ends (5′-RACE). Both classes of IGF-I mRNAs were expressed in a variety of tissues, with the highest level in liver; class 1 IGF-I mRNA was more abundant than class 2 IGF-I mRNA in all tissues. Six hours after a single intramuscular injection of 500 mg of recombinant bovine GH, class 1 and class 2 IGF-I mRNAs in steer liver were increased by 29% ( P=0.07) and 62% ( P<0.05), respectively. The luciferase reporter mRNA fused to a class 1 IGF-I 5′-untranslated region (5′-UTR) was translated four times more efficiently in vitro than the luciferase reporter mRNA fused to a class 2 IGF-I 5′-UTR ( P<0.05). These results indicate that the IGF-I gene in cattle is transcribed as class 1 and class 2 IGF-I mRNAs and that the two classes of IGF-I mRNAs may be regulated differentially at both transcriptional and translational levels.

Recombinant hemagglutinin protein of rinderpest virus expressed in insect cells induces cytotoxic T-cell responses in cattle.

Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.

Characterization of Shiga toxin producing E. coli and O157 serotype E. coli isolated in France from healthy domestic cattle

A study was carried out in France in collaboration with the meat industry to investigate the occurrence and characteristics of Shiga toxin-producing E. coli (STEC) and O157 E. coli in a population of healthy bovines representative of French livestock. A total of 851 animals belonging to three bovine classes (106 young bulls, 374 dairy cows and 371 meat cows) were included in the study. Samples of feces and of the corresponding carcasses were collected from March 97 to August 97 in seven abattoirs spread throughout the national territory. STEC cultures from the 1702 samples were screened using PCR for the presence of stx genes. Positive samples were further subjected to colony blot hybridization and to O157-specific immunomagnetic separation. Probe-positive colonies and O157 colonies were then analyzed for the presence of virulence genes and phenotypic characters (serotype, Stx production). In 154 (18.1%) feces and 91 (10.7%) carcass samples stx genes were detected. Two hundred and twenty-two STEC colonies were isolated from 67 (7.9%) feces and 16 (1.9%) carcass samples, with 183 STEC isolated from feces and 39 from carcasses. Only eight O157 isolates were collected from feces samples. None of these O157 E. coli isolates presented stx genes and thus could not be considered as pathogenic regarding hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). In 3.2% of STEC isolated from feces and in 10.2% of STEC from carcasses eae genes were detected. In 17% of STEC from feces and in 30.7% from carcasses ehx genes were detected. Using these data, the 222 STEC colonies could be classified in 11 different ‘virulence patterns’ (presence/absence of stx1, stx2, eae and ehx genes), showing that more than 77% of isolates presented only one virulence factor. Only three STEC on 222 colonies (1.3%) presented the three virulence factors stx, eae and ehx in association, none of them reacting with antisera specific for enterohemorrhagic E. coli. (EHEC). These data, together with the fact that only five isolates on the 222 (2.2%) reacted with such antisera (three O111 and two O26 isolates) demonstrated that the natural bacterial populations isolated during this study were clearly distinct from EHEC.

A Radiation Hybrid Map of BTA23: Identification of a Chromosomal Rearrangement Leading to Separation of the Cattle MHC Class II Subregions

Bovine chromosome 23 (BTA23) contains the bovine major histocompatibility complex (MHC) and is thus of particular interest because of the role of MHC genes in immunity. Previous studies have shown cattle MHC class II genes to be subdivided into two distinct subregions separated by a variable genetic distance of 15–30 cM. To elucidate the genetic events that resulted in the present organization of the class II and other MHC genes, a framework radiation hybrid (RH) map of BTA23 was developed by testing DNA samples from a 5000 rad whole genome RH panel. Twenty-six markers were screened with an average retention frequency of 0.27, ranging from 0.14 to 0.42. Total length of the chromosome was 220 cR5000, with 4.1 cR5000/cM when compared to linkage data. Gene orders for the markers common to both the RH framework map and the consensus framework linkage map are identical. Large centiray intervals,D23S23–D23S7, DYA–D23S24andCYP21–D23S31,were observed compared to linkage distances. These data may indicate a much larger physical distance or suppression of recombination in the interval separating the class II subregions and also within the class I region than previously estimated. Comparison of 13 Type I genes conserved between BTA23 and the human homolog HSA6p suggests the occurrence of an inversion encompassing the centromeric half of the bovine chromosome, thus explaining the large distance between the bovine class IIa and IIb clusters. These results exemplify the power of RH mapping in solving problems in comparative genomics and evolution. Furthermore, noncongruence of the genetic and physical RH map distances indicates that caution must be observed in using either resource alone in searching for candidate genes controlling traits of economic importance.

Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.

Regulation of MHC class I gene expression is at transcriptional and post-transcriptional level in bovine placenta

A previous study of MHC in cattle trophoblast demonstrated low or absent class I expression, using a broad specificity monoclonal antibody. The study reported here uses MHC-defined cattle and embryo transfer to ensure MHC incompatibility between dam and calf. Transcription and expression of defined class I genes was examined in placentomes taken at term, using monoclonal antibodies to bovine class I, a gene-specific DNA-based typing system, and in situ hybridisation. Results demonstrate intermediate levels of fetal MHC class I mRNA in trophoblast, but no detectable fetal class I protein. This suggests a level of transcriptional down-regulation, and a post-transcriptional block which might involve other gene products, such as β 2-microglobulin ( β 2m), or proteins involved in generation/transport of peptides.

Modulation of Bovine Leukemia Virus-Associated Spontaneous Lymphocyte Proliferation by Monoclonal Antibodies to Lymphocyte Surface Molecules

Both human T lymphotropic virus (HTLV) and bovine leukemia virus (BLV) infections are characterized byin vitroproliferation of peripheral blood lymphocytes in the absence of exogenous antigens or mitogens. Differential expression of lymphocyte surface molecules in HTLV and BLV infection suggests that lymphocyte dysregulation may involve signaling through surface molecules involved in immune regulation. We examined the expression of adhesion and major histocompatibility (MHC) molecules on circulating lymphocytes from BLV-infected cows with persistent lymphocytosis and the ability of monoclonal antibodies to these molecules to modulate spontaneous lymphocyte proliferation. The integrin molecule, CD11c, and both MHC class I and MHC class II molecules were upregulated on B and T lymphocytes from PL cows. Anti-CD11c antibody was stimulatory to lymphocyte proliferation regardless of BLV status and had a greater stimulatory effect on spontaneously proliferating lymphocytes from persistently lymphocytotic cows than on normal bovine lymphocytes. Antibodies to bovine class I and class II inhibited spontaneous lymphocyte proliferation. Results suggest that lymphocyte dysregulation in BLV-induced persistent lymphocytosis involves upregulation of and signaling through lymphocyte surface molecules which are involved in immune activation of lymphocytes.

Localization of bovine lymphocyte antigen (BoLA) DYA and class I loci to different regions of Chromosome 23

The class II loci of the major histocompatibility complex (MHC)of humans (HLA) and mice (H-2) are embedded among other lociof the MHC, which spans approximately 4 mb of DNA and has arecombination length of about 2 cM (Campbell and Trowsdale1993; Himmelbauer et al. 1993).In contrast, recombination analysis of class II genes in thebovine lymphocyte antigen complex (BoLA) revealed two distinctclusters of class II loci about 17 cM apart (Anderson et al. 1988).One cluster designated IIa contains the bovine homologs of thehuman DQ and DR gene families and is tightly linked to otherBoLA genes, including HSP70, CYP21, and several class I loci(Bishop et al. 1994). The second cluster, IIb, contains the DYA,DYB, DOB loci and a novel class II gene designated DIB (Stoneand Muggli-Cockett 1990). The disruption of the BoLA class IIloci into two clusters suggests that the BoLA complex has under-gone significant rearrangements not yet observed in the MHCs ofhumans or mice, but that apparently have occurred in the MHCs ofchickens (Miller et al. 1994) and swine (Smith et al. 1995). Alter-natively, the bovine class IIa and IIb gene clusters could be inphysical proximity but undergo frequent recombinant owing to oneor more recombination hotspots. Recombination hotspots havebeen reported in the HLA and H-2 regions (Shiroishi et al. 1990;Steinmetz et al. 1986; Yoshino et al. 1994), and anomalous re-combination frequencies in this region among individual bullshave been reported (Park et al. 1995).To test whether the BoLA IIb cluster mapped to a differentchromosomal location than the other BoLA loci, we performedfluorescence in situ hybridization (FISH) analysis of bovine Chro-mosome (Chr) 23, using large insert clones containing DYA orclass I sequences as fluorescent probes. In this report, we demon-strate that DNA probes containing bovine DYA sequences map toa different region of Chr 23 than does DNA containing class Isequences, consistent with the reported recombination distance of17 cM between these genes.Recombinant clones of bacterial artificial chromosomes(BACs) containing DYA or class I sequences were isolated byPCR screening of a bovine BAC library (Cai et al. 1995) withprimer pairs that have been previously described (Van Eijk et al.1992; Garber et al. 1994). Authenticity of each clone was con-firmed by sequencing of the respective PCR products and com-paring with published sequences.BAC clones 6 and 76 were identified by PCR as containingDYA and class I sequences, respectively, and DNAs from theseclones were prepared for FISH. The sequence of the PCR productfrom BAC 6 differed at only a single position,

Nucleotide Sequences and the Molecular Evolution of the DMA and DMB Genes of the Bovine Major Histocompatibility Complex

cDNA clones encoding the bovine major histocompatility complex (MHC) class II DM α- and β-chains were isolated and characterized. The BoLA-DMA cDNA clone, MA7, encoded a primary translated product of 260 amino acids, which included a signal peptide of 26 amino acids and a mature polypeptide of 234 amino acids. The BoLA-DMB cDNA clone, MB6, encoded a primary translated product of 262 amino acids, with a signal peptide of 18 amino acids and a mature polypeptide of 244 amino acids. Comparison of the sequences and construction of a phylogenetic tree revealed that both clones are more closely related to human and mouse DM genes than to genes for conventional bovine class II α- and β-chains. Thus, since the bovine DMA and DMB genes are so different from other class II sequences and show evidence of strong conservation (>70%) among the bovine, mouse and human homologues, it seems likely that each of these cDNA clones encodes a functional product, which might perform an important function, as previously established in studies in mouse and man.

An assay for the identification of antigens recognized by cytotoxic T cells, based on transient transfection of COS cells

The studies reported here describe methodology permitting the direct identification of antigens recognized by cytotoxic T lymphocytes. We demonstrated that bovine alloreactive CTL can detect a bovine MHC molecule transiently expressed in a COS cell population in a standard microcytotoxicity assay. We then showed that alloreactive CTL can detect cells expressing the bovine class I MHC molecule in a population of cells transfected with the plasmid containing the corresponding gene plus 100-fold as many plasmids containing an irrelevant gene. In addition, the transiently transfected COS cells can specifically restimulate CTL as detected by a standard microcytotoxicity assay using the target cell line. Overall, the results suggest that COS cells could be employed for the direct screening of an antigen or antigen gene library by immune CTL.