Association of the bovine leukocyte antigen major histocompatibility complex class II DRB3*4401 allele with host resistance to the Lone Star tick, Amblyomma americanum

The MHC of cattle, known as the bovine leukocyte antigen (BoLA) complex, plays an integral role in disease and parasite susceptibility, and immune responsiveness of the host. While susceptibility to tick infestation in cattle is believed to be heritable, genes that may be responsible for the manifestation of this phenotype remain elusive. In an effort to analyze the role that genes within the BoLA complex may play in host resistance to ticks, we have evaluated components of this system within a herd of cattle established at our laboratory that has been phenotyped for ectoparasite susceptibility. Of three microsatellite loci within the BoLA complex analyzed, alleles of two microsatellite loci within the BoLA class IIa cluster (DRB1-118 and DRB3-174) associated with the tick-resistant phenotype, prompting further investigation of gene sequences within the DRB3 region. DRB3 is a class IIa gene, the second exon of which is highly polymorphic since it encodes the antigen recognition site of the DR class II molecule. Analysis of the second exon of the DRB3 gene from the phenotyped calves in our herd revealed a significant association between the DRB3*4401 allele and the tick-resistant phenotype. To our knowledge, this is the first report of a putative association between a class IIa DRB3 sequence and host resistance to the Lone Star tick. Elucidation of the mechanism involved in tick resistance will contribute to improving breeding schemes for parasite resistance, which will be beneficial to the cattle industry.     

哈萨克羊DRB1基因外显子3多态性及生物信息学分析

试验旨在研究白细胞表面抗原DRB1基因外显子3多态性与哈萨克羊布鲁氏菌病易感性的相关性。运用混合DNA池结合PCR产物直接测序方法,对哈萨克羊DRB1基因外显子3进行多态性分析,经卡方检验分析每个SNP位点的等位基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性,利用生物信息学分析软件对PCR扩增所获序列进行RNA二级结构及蛋白质的二级结构和抗原表位分析。结果表明,在282 bp的外显子3序列中共检测到7个SNPs,分别为：T10C、C119T（Trp→Arg）、G215C（Gln→Glu）、A238G、T245G（Ser→Ala）、G256A、C259T,这些位点在病例组和对照组之间的等位基因频率及各基因型间不存在显著性差异（P > 0.05）;进一步分析发现,各突变位点均引起RNA二级结构和最小自由能的改变,各错义突变位点均未引起蛋白质二级结构和抗原表位的改变。由此得出,DRB1基因外显子3的7个SNPs位点（T10C、C119T、G215C、A238G、T245G、G256A和C259T）与哈萨克羊布鲁氏菌病易感性无相关性。     

Association of BoLA-DRB3 alleles identified by a sequence-based typing method with mastitis pathogens in Japanese Holstein cows

The association of the polymorphism of bovine leukocyte antigen ( BoLA-DRB3 ) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101 , *1501 , *1201 , and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101 , *1501 , *1201 , and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli , and DRB3*1501 might be associated with susceptibility to Escherichia coli . However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci , and DRB3*1201 , with resistance to Streptococci , Escherichia coli , and Staphylococcus aureus .     

Association of BoLA‐DRB3 alleles with mastitis resistance and susceptibility in Japanese Holstein cows

In this study, 714 cows from 26 dairy herds were reclassified as healthy or mastitic cows on the basis of long‐term somatic cell count (SCC) in milk. Cows with more than three consecutive lactation records of SCC from the first or second to fifth lactation, were selected, and their BoLA‐DRB3 (DRB3) alleles were identified using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. Cows with an SCC of < 200 000 cells/mL in all monthly records were classified as healthy (n = 91). Cows with an SCC of > 300 000 cells/mL in two consecutive tests or four non‐consecutive tests or cows with an SCC of > 500 000 cells/mL in any one test during lactation, regardless of parity, were classified as mastitic (n = 201). Mastitic cows (n = 153) from another 40 herds were considered to be infected if bacteriological testing revealed mastitis pathogens in milk. Their DRB3 alleles were identified using PCR‐sequence‐based typing (PCR‐SBT). The differences in DRB3 allelic frequencies between healthy cows and cows with various degrees of mastitis were re‐investigated. Moreover, the associations of various amino acid motifs in DRB3 alleles with resistance or susceptibility to mastitis pathogens were re‐examined. DRB3.2*8(DRB3*1201) and DRB3.2*16(DRB3*1501) alleles were found to be associated with susceptibility, while DRB3.2*22(DRB3*1101), DRB3.2*23(DRB3*2703), and DRB3.2*24(DRB3*0101) alleles were found to be associated with resistance.     

Analysis of BoLA class II microsatellites in cattle infested with Boophilus microplus ticks: class II is probably associated with susceptibility

The aim of this study was to determine the role of certain bovine lymphocyte antigens (BoLA) regions in the resistance or susceptibility to Boophilus microplus tick infestation in two different breeds of cattle. The breeds were maintained, one in natural conditions and the second one in an experimental setting at the research station in Martinez de la Torre, Veracruz, Mexico. The study took place from June to August 2001 (natural infestation) using 33 crossbreed steers (crossbreed is here defined as 3/4 European = 1/2 Simmenthal x 1/4 Holstein x 1/4 Zebu, a cross resulting from F1 x Simmenthal), ranging from 15 to 20 months old. Fifty-nine F1 cows (1/2 Holstein x 1/2 Zebu) were included in the experimental setting, infested and followed during 25 days in November 2001 and 2002. Experiment A included thirty-one 2-7-year-old F1 cows, and experiment B included twenty-eight 18-24-month-old F1 heifers. Both groups were analysed separately and were not comparable because of the different infestation methods and genetic background. All ticks > or =4mm long were counted on the total body of F1 animals and on one side of the 3/4 European steers. In this case, susceptible animals were defined when having ticks = X + 1S.D. (29 +/- 16). In the experimental setting susceptibility was defined when the number of ticks was over the 75 percentile (> or =79). DNA was extracted from peripheral blood samples of all animals. The BoLA DRB3, DRBP1, RM185 and BM1815 microsatellite loci were amplified using a PCR method. Genescan software was used for analysis in an ABI sequencer. The SPSS statistical program was used and the comparisons were assessed using the Fisher's exact test. In the naturally infested animals, DRB3-184 was found positively associated with tick infestation (P = 0.018; Pc = NS; OR = 5; EF = 28%). DRBP1-128 was also found to be increased (P = 0.03; Pc = NS; OR = 6; EF = 42%). In the experimentally infested animals, two more loci were found to be associated, BM1815-152 (P = 0.01; Pc = NS; OR = 15; EF = 74%) and DRBP1-130 (P = 0.05; Pc = NS; OR = 4; EF = 77%). None of them remained significant after correction, indicating that a larger sample size is needed to confirm the results. This is the first study showing MHC genes associated with tick infestation based on class II microsatellite polymorphisms. Further studies are needed to confirm the susceptibility traits and to determine haplotype segregation in families.     

从江香猪SIM1基因SNPs筛查及生物信息学分析

本研究以从江香猪为研究对象,二元杂交猪(从江香猪×野猪)、外三元杂交猪(杜×长×大)为对照,采用DNA池和直接测序技术对3个群体的SIM1基因7个外显子、部分内含子以及3'非翻译区序列进行多态性检测;利用生物信息学软件预测多态位点对SIM1基因mRNA二级结构和蛋白质一级、二级结构的影响。结果表明,在3个群体的SIM1基因中筛查到12个SNPs,C77T位于第5外显子,T29186C、A29195C位于第9内含子,C63T、C225T位于第10外显子,C107T、A426G、T583C、A586C、A605C、A615C位于第11外显子,G267T位于3'非翻译区。其中C77T、T29186C、A29195C、C63T、C225T、C107T、G267T为同义突变,A426G、T583C、A586C、A605C、A615C为错义突变;5个错义突变分别导致异亮氨酸(Ile)变为缬氨酸(Val)、亮氨酸(Leu)变为脯氨酸(Pro)、谷氨酸(Glu)变为丙氨酸(Ala)、谷氨酸(Glu)变为天冬氨酸(Asp)、天冬酰胺(Asn)变为组氨酸(His);根据在线软件预测,突变前后的SIM1基因mRNA二级结构和蛋白质一级、二级结构均会发生改变。     

SNPs Screening and Bioinformatics Analysis of SIM1 Gene in Congjiang Pigs

In this study, Congjiang pig was served as a research object, crossbred pigs (Congjiang pig×boar), outside crossbred pigs (Duroc×Landrace×Yorkshire) as controls.Using DNA pools and direct sequencing detected polymorphism of 7 exons, part of introns and 3'untranslated region (3'-UTR) sequences of SIM1 gene for three groups.Bioinformatics software predicted what impact polymorphic loci had to mRNA secondary structure and protein primary, secondary structure of SIM1 gene.The results showed that 12 SNPs were screened in SIM1 gene of three groups, C77T located in exon 5, T29186C and A29195C located in intron 9, C63T and C225T located in exon 10, and C107T, A426G, T583C, A586C, A605C and A615C located in exon 11, G267T located in 3'-UTR. C77T, T29186C, A29195C, C63T, C225T, C107T and G267T were silent mutations, A426G, T583C, A586C, A605C and A615C were missense mutations.The five missense mutations respectively led to isoleucine (Ile) into valine (Val), leucine (Leu) into proline (Pro), glutamate (Glu) into alanine (Ala), glutamic (Glu) into aspartic (Asp), asparagine (Asn) histidine (His), and according to online software forecast, mRNA secondary structure and protein primary, secondary structure of SIM1 gene changed in mutations before and after.     

Analysis of Relationship between Bovine Lymphocyte Antigen DRB3.2 Alleles, Somatic Cell Count and milk Traits in Iranian Holstein Population

The major histocompatibility complex (MHC) is a gene complex closely linked to the vertebrate immune system due to its importance in antigen recognition and immune response to pathogens. To improve our understanding of the MHC and disease resistance in dairy cattle, we gathered 5119 test day records of somatic cell count (SCC) and performance traits of 262 Holstein dairy cows to determine whether the DRB region of the MHC contains alleles that are associated with elevated SCC, milk yield, protein and fat percent of milk. To this purpose, genotyping of animals for DRB3 gene was investigated by polymerase chain reaction‐based restriction fragment length polymorphism (PCR‐RFLP) assay. A two‐step PCR was carried out so as to amplify a 284 base‐pair fragment of exon 2 of the target gene. Second PCR products were treated with three restriction endonuclease enzymes RsaI, BstYI and HaeIII. Twenty‐eight BoLA‐DRB3 alleles were identified including one novel allele (*40). The results in general are in good accordance with allele frequencies of Holstein cattle populations reported by previous studies. Analyses of associations were modeled based on repeated measurement anova and generalized logistic linear methods for production traits and SCC data, respectively. The results of this study showed a significant relationship between the elevated SCC reflecting an increased probability of occurrence to subclinical mastitis and DRB3.2 allele *8 (p < 0.03). The results also revealed significant positive relationships of alleles*22 (p < 0.01) and allele*11 (p < 0.05) with milk fat percent as well as of alleles*24 (p < 0.03) and *22 (p < 0.05) with protein percent. The present study failed to find any association between milk yield and tested alleles. Because of the lack of consistency among results of similar studies, we suggest further investigations to determine the precise nature of these associations with the high polymorphic bovine MHC region to be performed based on haplotypes.     

Analysis of host genetic factors influencing African trypanosome species infection in a cohort of Tanzanian Bos indicus cattle

Trypanosomosis caused by infection with protozoan parasites of the genus Trypanosoma is a major health constraint to cattle production in many African countries. One hundred and seventy one Bos indicus cattle from traditional pastoral Maasai (87) and more intensively managed Boran (84) animals in Tanzania were screened by PCR for the presence of African animal trypanosomes (Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei), using blood samples archived on FTA cards. All cattle screened for trypanosomes were also genotyped at the highly polymorphic major histocompatibility complex (MHC) class II DRB3 locus to investigate possible associations between host MHC and trypanosome infection. Overall, 23.4% of the 171 cattle tested positive for at least one of the three trypanosome species. The prevalence of individual trypanosome species was 8.8% (T. congolense), 4.7% (T. vivax) and 15.8% (T. brucei). The high prevalence of T. brucei compared with T. congolense and T. vivax was unexpected as this species has previously been considered to be of lesser importance in terms of African bovine trypanosomosis. Significantly higher numbers of Maasai cattle were infected with T. brucei (23.0%, p=0.009) and T. congolense (13.8%, p=0.019) compared with Boran cattle (8.3% and 3.6%, respectively). Analysis of BoLA-DRB3 diversity in this cohort identified extensive allelic diversity. Thirty-three BoLA-DRB3 PCR-RFLP defined alleles were identified. One allele (DRB3*15) was significantly associated with an increased risk (odds ratio, OR=2.71, p=0.034) of T. brucei infection and three alleles (DRB3*35, *16 and *23) were associated with increased risk of T. congolense infection. While further work is required to dissect the role of these alleles in susceptibility to T. brucei and T. congolense infections, this study demonstrates the utility of FTA archived blood samples in combined molecular analyses of both host and pathogen.     

The Correlation Analysis between Chinese Merino Sheep OLA-DQB1 Gene Exon 2 Genetic Polymorphism and Brucellosis Susceptibility

The single nucleotide polymorphisms (SNPs) of ovine lymphocyte antigen DQB1 (OLA-DQB1) gene exon 2 was amplified by PCR-SSCP method from 148 healthy and 60 infected with Brucella Chinese Merino sheep and then PCR products of different alleles were sequenced to determine the polymorphism loci of the gene.The differences in gene frequency and genotype frequency of each SNP loci were analyzed statistically to analyze its correlation with brucellosis susceptibility.The sequencing result showed that 43 SNPs were detected in 270 bp DNA sequence,the gene frequencies of G196A allele had extremely significant difference in case and control samples (P< 0.01),and its genotype frequencies presented significant difference (P< 0.05).Similarly,C211T allele was significantly different in case and control samples (P< 0.05).The results showed that the polymorphism of OLA-DQB1 gene exon 2 might be a significant association gene with brucellosis susceptibility.     

中国美利奴羊OLA-DQB1基因exon2遗传多态性与布鲁氏菌病易感性的关联分析

本试验采用PCR-SSCP方法对148只布鲁氏菌阴性和60只布鲁氏菌阳性中国美利奴羊白细胞表面抗原DQB1(OLA-DQB1)基因exon 2单核苷酸多态性(SNPs)进行了检测,之后挑选不同等位基因进行PCR产物测序,旨在确定该基因的多态性位点,并对每个SNP位点的等位基因频率、基因型频率进行统计分析,从而分析其多态性与布鲁氏菌病易感性的相关性.测序结果表明,在270 bp的序列内共检测到43个SNPs,其中G196A位点的等位基因频率在病例组和对照组中的分布存在极显著差异(P< 0.01),其基因型频率存在显著差异(P< 0.05);C211T位点的等位基因频率在病例组和对照组中存在显著差异(P< 0.05).由此表明,OLA-DQB1基因exon 2多态性与中国美利奴羊布鲁氏菌病易感性呈显著相关.     

Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates

Ninety-five avian pathogenic Escherichia coli (APEC) isolates recovered from diagnosed cases of avian colibacillosis from North Georgia between 1996 and 2000 were serotyped and examined for typical virulence-factors, susceptibility to antimicrobials of human and veterinary significance, and genetic relatedness. Twenty different serotypes were identified, with O78 being the most common (12%). The majority of the avian E. coli isolates (60%), however, were non-typeable with standard O antisera. Eighty-four percent of isolates were PCR positive for the temperature-sensitive hemagglutinin (tsh) gene and 86% positive for the increased serum survival (iss) gene. Multiple antimicrobial-resistant phenotypes (> or =3 antimicrobials) were observed in 92% of E. coli isolates, with the majority of isolates displaying resistance to sulfamethoxazole (93%), tetracycline (87%), streptomycin (86%), gentamicin (69%), and nalidixic acid (59%). Fifty-six E. coli isolates displaying resistance to nalidixic acid were co-resistant to difloxacin (57%), enrofloxacin (16%), gatifloxacin (2%), and levofloxacin (2%). DNA sequencing revealed point mutations in gyrA (Ser83-Leu, Asp87-Tyr, Asp87-Gly, Asp87-Ala), gyrB (Glu466-Asp, Asp426-Thr), and parC (Ser80-Ile, Ser80-Arg). No mutations were observed in parE. Twelve of the quinolone-resistant E. coli isolates were tolerant to cyclohexane, a marker for upregulation of the acrAB multi-drug resistance efflux pump. Quinolone-resistant isolates were further genetically characterized via ribotyping. Twenty-two distinct ribogroups were identified, with 61% of isolates clustering into four major ribogroups, indicating that quinolone resistance has emerged among multiple avian pathogenic E. coli serogroups and chromosomal backgrounds.     

哈萨克羊DQB2基因外显子2多态性及遗传变异分析

试验旨在研究DQB2基因外显子2多态性与哈萨克羊布鲁氏菌病易感性的相关性。通过PCR-SSCP技术对146只布鲁氏菌阴性哈萨克羊血液样本和28只布鲁氏菌阳性哈萨克羊血液样本中的白细胞表面抗原DQB2基因外显子2的多态性进行研究,挑取不同的等位基因进行克隆测序,经卡方检验分析每个SNP位点的基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性,应用生物信息学软件分析与哈萨克羊布鲁氏菌病易感性相关的不同等位基因的mRNA二级结构及蛋白质的二级结构、三级结构和抗原表位。结果发现,在270 bp的外显子2序列中共检测到33个SNPs,其中C9G、A180G位点的基因频率在病例组和对照组中的分布具有极显著性差异（P<0.01）,其基因型频率在病例组和对照组中存在显著差异（P<0.05）;A13T、C133G位点的基因频率在病例组和对照组中存在显著差异（P<0.05）;进一步分析发现,A180G突变位点的最小自由能最低,其mRNA二级结构最稳定;A13T和C133G 2个突变位点均引起mRNA二级结构及蛋白质二级结构、三级结构和抗原表位的改变。本试验结果表明DQB2基因外显子2多态性与哈萨克羊布鲁氏菌病易感性呈显著相关。     

Polymorphisms and Its Genetic Variation Analysis of DQB2 Gene Exon 2 in Kazakh Sheep

This study was aimed to investigate the association between the polymorphism of DQB2 gene exon 2 and the susceptibility to Kazakh sheep brucellosis. The DQB2 gene exon 2 of Kazakh sheep lymphocyte antigen was amplified by PCR-SSCP method from 146 healthy and 28 infected with Brucella Kazakh sheep, and the single nucleotide polymorphisms (SNP) was analyzed, then the different alleles were selected for cloning and sequencing. In order to analyze its correlation with brucellosis susceptibility, the differences in gene frequency and genotype frequency of each SNP locus were analyzed by Chi-square test. Bioinformatics softwares were used to analyze the secondary structure of mRNA, the secondary structure, tertiary structure and epitope of protein. The sequencing result showed that 33 SNPs were detected in 270 bp DNA sequence, the gene frequencies of C9G and A180G were extremely significantly different in case group and control group (P<0.01), and its genotype frequencies presented significantly difference (P<0.05). Similarly, A13T and C133G loci were significant difference in case group and control group (P<0.05). Further analysis result showed that the minimum free energy of the A180G mutation site was the lowest and its mRNA secondary structure was the most stable; Both A13T and C133G mutation sites caused the changes of mRNA secondary structure, protein secondary, tertiary structure and antigenic epitope of protein, respectively. The results showed that the polymorphism of DQB2 gene exon 2 might be significantly correlated with brucellosis susceptibility in Kazakh sheep.     

Exposure of young dairy cattle to Mycobacterium avium subsp. paratuberculosis (MAP) through intensive grazing of contaminated pastures in a herd positive for Johne’s disease

This study investigated the susceptibility of 1- to 2-year-old cattle to Mycobacterium avium subsp. paratuberculosis (MAP) on pasture previously grazed by infected cattle. The exposure of yearling cattle to pastures contaminated with MAP resulted in infection with MAP, showing that age resistance to infection can be overcome by pressure of infection.     

Study on the association of BoLA-DRB3.2 alleles with clinical mastitis in Norwegian Red cows

Genotyping of bovine leucocyte antigen DRB3.2 (BoLA-DRB3.2) in a total of 523 Norwegian Red (NR) cows from two groups selected for high protein yield and low clinical mastitis, respectively, identified 27 previously reported BoLA-DRB3.2 alleles across the groups. Significant differences in BoLA-DRB3.2 allele frequencies were found between the selection groups. Alleles *13, *18, *22 and *27 had a significantly higher frequency in cows selected for low clinical mastitis, while alleles *3, *9, *11 and *26 had a higher frequency in cows selected for high protein yield. Associations between BoLA-DRB3.2 alleles and clinical mastitis were analysed based on mastitis data from 741,072 first-lactation NR cows, of which 452 were genotyped. Alleles *22 and *26 were found to be associated with increased clinical mastitis, while alleles *7, *11, *18 and *24 had a favourable effect on mastitis resistance. Contradictory results from different studies investigating associations between BoLA-DRB3.2 alleles and mastitis indicate that future studies should focus on associations of mastitis with BoLA haplotypes rather than with single BoLA genes.     

Evaluation of the immunogenicity of Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins

The aim of this study was to assess the humoral immune responses in MAP-infected and uninfected sheep against 27 MAP stress-associated recombinant proteins that were regulated in in vitro models of physiological stress. These include evaluation of 5 proteins, which were previously reported by Gumber et al. (2009b), using serum samples from sheep with a wide range of disease stages. For purification of recombinant his-tagged proteins expressed as an insoluble protein, on-column refolding purification was applied as well as one-step denaturing purification. All purifications together resulted in a total of 48 recombinant antigen preparations. In antibody ELISA tests, 23 of these, representing 18 MAP proteins, showed significant differences in responses between infected and uninfected sheep. Recombinant antigen preparations MAP2281c, MAP3555 (refolded form), and MAP0711c (refolded form) when incorporated in an ELISA, had similar sensitivity to a commercial antibody ELISA test at the cutpoint of 90% specificity, and showed relatively high values in receiver operating characteristic (ROC) curve analysis. However, as some of the sera from uninfected sheep also reacted to recombinant antigens, further development of the assays is necessary prior to practical application. Compared to the commercial antibody ELISA, MAP0593c, MAP2281c, MAP2411, MAP3555, and MAP3200 detected more infected sheep with a lower grade of lesion, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected sheep at an early stage of infection. This is consistent with the hypothesis of latency or dormancy in subclinical mycobacterial infection.     

Altered patterns of toll-like receptor gene expression in cull cows infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease of cattle. The mechanism how MAP can co-exist in the gastro-intestinal tract despite a massive infiltration of immune cells is not known. Toll-like receptors (TLRs) are known to play an important role in both innate and acquired immune responses but it is unclear what role different TLRs play in response to MAP. In this study, 38 cull cows from herds infected with MAP were classified into four groups, based on MAP culture from gut tissues and histopathological lesion scores. The expression of TLR1, 2 and 4 mRNA from MAP antigen-stimulated mesenteric lymph node (MLN) cultures and peripheral blood mononuclear cells (PBMCs) and in the MLN and ileum tissues of these animals was determined. MAP antigen-specific expression of TLR1 in MLN and PBMC was significantly lower in the MAP-infected groups than the non-infected control group, suggesting that in MAP-infected animals there is impairment in the up-regulation of TLR1 in response to MAP antigen. TLR4 expression in MLN tissues was significantly higher in the severely infected group than the control group suggesting up-regulation of endogenous TLR4 expression at a site of MAP infection in animals severely affected with Johne's disease. A preliminary screening of TLR1, 2 and 4 in the cull cows revealed the presence of polymorphisms in TLR1 and TLR2. In summary, one mechanism how MAP may subvert the immune system is that there is an apparent lack of recognition of MAP antigens as foreign by TLR1 in MAP-infected cows.     

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.