Cytokine profiles in chickens infected with virulent and avirulent Marek's disease viruses: Interferon-gamma is a key factor in the protection of Marek's disease by vaccination

Marek's disease has been controlled by vaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-γ induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-γ could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.     

Mapping of susceptibility to Marek's disease within the major histocompatibility (B) complex by refined typing of White Leghorn chickens

The major histocompatibility (B) complex of a distinct commercial pure White Leghorn chicken line was characterized using serological, biochemical and restriction fragment length polymorphism (RFLP) typing. Line B chickens displayed a high recombination frequency within the B complex. Three recombinant haplo-types were identified. The influence of these haplotypes was determined in relation to the haplotypes B^l9 and B²¹ on their resistance to Marek's disease (MD) in an experimental infection with the virus. Offspring of sires with a recombinant haplotype in combination with B¹⁹ or B²¹, and dams, which were homozygous B¹⁹/B¹⁹ or B²¹/B²¹ were infected. The B type of the offspring had a significant effect upon survival. Animals with B complex types B²¹/B²¹, B¹³⁴/B²¹ and B²³⁴/B²¹ were relatively resistant to MD (24–32% mortality), whereas B¹⁹/B¹⁹ birds were highly susceptible (68% mortality). Animals with a recombinant halpotype B^19r21 (B-G²¹, B-F¹⁹) were equally susceptible to MD as birds with the complete B¹⁹ haplotype. In contrast to earlier publications, resistance was not inherited as a dominant trait. Apparently, B¹⁹ was associated with a dominant susceptibility. The gene(s) associated with the B complex and involved in resistance to MD were localized within the B-F/B-L region. However, the association with a presumably non-coding subregion of B-G could not be excluded.     

High resolution mapping and identification of new quantitative trait loci (QTL) affecting susceptibility to Marek's disease

Marek's disease (MD) is a lymphoproliferative disease of chickens that costs the poultry industry approximately $1 billion annually. Genetic resistance to MD is gaining increased attention to augment vaccinal control as disease outbreaks occur more frequently. Previously, analysis of a 272 F2 White Leghorn resource population measured for many MD traits and genotyped for 78 microsatellite markers revealed two and four quantitative trait loci (QTL) with significant and suggestive association, respectively, to one or more MD associated traits. Additional genetic markers have since been scored on the MD resource population to increase QTL resolution and genome coverage. Saturation of four of the QTL regions with 17 markers revealed five new QTL while 32 markers extended the genome coverage by 400 + CM and uncovered three more QTL. QTL analysis by single-point and interval mapping algorithms agreed well when marker saturation was approximately 20 CM or less. Currently 127 genetic markers cover approximately 68% of the genome that contain up to 14 MD QTL associated to one or more MD trait; seven at the significant level and seven at the suggestive level. Individually each QTL accounts for 2-10% of the variation and, in general, resistance was dominant although the resistant allele may come from either parental line. This study suggests that a limited number of genomic regions play a major role in the genetic control of MD resistance. Markers linked to these loci may be useful for selection of MD resistant stock by the poultry industry following verification of the association within their breeding populations.     

Mapping quantitative trait loci for binary traits using a heterogeneous residual variance model: an application to Marek's disease susceptibility in chickens

A typical problem in mapping quantitative trait loci (QTLs) comes from missing QTL genotype. A routine method for parameter estimation involving missing data is the mixture model maximum likelihood method. We developed an alternative QTL mapping method that describes a mixture of several distributions by a single model with a heterogeneous residual variance. The two methods produce similar results, but the heterogeneous residual variance method is computationally much faster than the mixture model approach. In addition, the new method can automatically generate sampling variances of the estimated parameters. We derive the new method in the context of QTL mapping for binary traits in a F2 population. Using the heterogeneous residual variance model, we identified a QTL on chromosome IV that controls Marek's disease susceptibility in chickens. The QTL alone explains 7.2% of the total disease variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

马立克氏病病毒超强毒感染鸡羽髓蛋白质组分析

【目的】羽毛是细胞游离马立克氏病病毒(Marek’s disease virus,MDV)释放的部位,为了解感染MDV后鸡羽中宿主基因表达的变化及对病毒感染的应答,进行了MDV感染鸡的羽髓蛋白质组学分析。【方法】1日龄无特定病原体(specific pathogen free,SPF)鸡人工感染MDV超强毒RB1B株(1000PFU),感染后21d采集鸡羽毛,提取羽髓蛋白,以17cm,pH5-8的IPG胶条进行二维电泳,以未感染病毒的SPF鸡羽髓蛋白为对照,使用PDQuest软件对二维电泳图谱进行差异蛋白分析,并选取部分差异斑点进行质谱鉴定。【结果】PDQuest软件分析发现攻毒组和对照组表达差异大于两倍的蛋白点有41个,其中攻毒组表达上调的蛋白点25个,下调的蛋白点7个,新出现的蛋白点有9个。质谱分析共成功鉴定了21个斑点,对应于20个蛋白。如载脂蛋白AI(apolipoprotein AI)、14-3-3 sigma(两个斑点均为该蛋白)、癌蛋白18(stathmin)等。【结论】功能预测表明这些蛋白涉及到宿主的抗病毒应答、物质代谢、细胞骨架成分、细胞增殖相关等方面。     

敲除meq的鸡马立克氏病毒强毒株对超强毒的免疫保护作用

【目的】比较和评价一株敲除了meq基因的马立克氏病毒(MDV)的致病性及其诱发的保护性免疫作用。【方法】将1日龄SPF鸡150只随机分为5组,每组30只,分别饲养于正压过滤空气的SPF动物饲养隔离罩内。1日龄时,第1和第5组鸡以2000PFU/只的剂量腹腔接种GX0101Δmeq,第2组鸡以2000PFU/只的剂量腹腔接种CVI988/Rispens疫苗株,第3和第4组鸡不接种任何病毒作为对照组。免疫接种5d后,第1、2、3组分别以500PFU/只的剂量攻击MDV超强毒株vvrMd5。饲养90d,观察死亡情况,对各组死亡鸡只剖检,并取疑似马立克特有病变脏器做石蜡切片,于攻毒后90d处死全部存活鸡并随机取心脏、肝脏、脾脏做病理切片。【结果】单独接种GX0101Δmeq的第5组没有任何马立克氏病临床症状和特有的组织学病变,接种GX0101Δmeq再感染超强毒株vvrMd5的第1组也没有马立克病特有的组织学病变,但CVI988/Rispens免疫后感染超强毒株vvrMd5的第2组显示马立克病特有病变的病理切片比例为9/42,单独接种超强毒株vvrMd5的第3组死亡率为87%,死亡鸡出现可眼观典型肿瘤率为25%,免疫接种GX0101Δmeq和CVI988/Rispens的第1组和第2组对超强毒株vvrMd5攻击的保护指数分别为100%和89%。【结论】本实验构建的MDVmeq基因缺失株-GX0101Δmeq可在体外稳定复制,不仅对SPF鸡没有致病性和致瘤性,而且能诱导比CVI988/Rispens疫苗株更好的对超强毒MDV的免疫保护效果。     

Random amplified polymorphic DNA markers in crosses between inbred lines of Rhode Island Red and White Leghorn chickens

Reciprocal crosses and backcrosses were conducted between inbred Rhode Island Red and White Leghorn chickens differentiated for egg production and egg quality traits. Random amplified polymorphic DNA (RAPD) markers distinguishing inbred lines were detected. Twenty-two polymorphic bands were found from screening 120 single 10-mer random primers of which two were consistent with sex-linked markers. Of 90 pairwise two-point linkage analyses completed for the autosomal markers, four close linkages (8·2 cM to 14·9 cM) were significantly different from zero.     

利用线粒体COI基因揭示中国乌骨鸡遗传多样性和群体遗传结构

全面了解中国乌骨鸡的遗传背景有利于保护和开发利用其种质资源。本研究测定了中国12个乌骨鸡品种线粒体细胞色素c氧化酶亚基I (cytochrome c oxidase subunit I, COI)基因, 比较分析其遗传多样性和群体遗传结构。255份乌骨鸡样品共检测到22个变异位点, 占分析位点的3.17%; 核苷酸多样性为0.00142-0.00339, 单倍型多样性为0.380-0.757, 其中略阳乌鸡核苷酸多样性最高, 德化黑鸡最低。检测到7个氨基酸变异位点, 来自6个品种共11个个体。定义了24种单倍型, 其中单倍型H1和H3为12个乌骨鸡品种共享, 出现频率分别为115次和64次; 盐津乌骨鸡单倍型数最多, 广西乌鸡最少。中性检验与错配分析显示实验种群未经历显著的群体扩张事件。分子变异分析显示81.06%的变异来自群体内; 品种间遗传距离为0.002-0.004, 品种间遗传分化系数F_st值为-0.035至0.594, 雪峰乌骨鸡与其他种群间的遗传分化程度最高。邻接树显示, 乌骨鸡未能独立形成分支, 不能从家鸡和红原鸡中有效区分开来。中国乌骨鸡中介网络图将24个单倍型分为3条进化主支, 呈现出一定的品种特异性, 由无量山乌骨鸡、云南盐津乌骨鸡和雪峰乌骨鸡组成单倍型H8、H9、H11、H12游离于这3条进化主支之外。增加其他家鸡和红原鸡COI基因的中介网络图主体结构与中国乌骨鸡的相同。结果表明中国乌骨鸡品种遗传多样性较低, 但品种间遗传分化显著, 可能是从当地家鸡中选育而来, 需要加强种质资源的保护。     

A strategy to identify positional candidate genes conferring Marek''s disease resistance by integrating DNA microarrays and genetic mapping

Marker-assisted selection (MAS) to enhance genetic resistance to Marek's disease (MD), a herpesvirus-induced T cell cancer in chicken, is an attractive alternative to augment control with vaccines. Our earlier studies indicate that there are many quantitative trait loci (QTL) containing one or more genes that confer genetic resistance to MD. Unfortunately, it is difficult to sufficiently resolve these QTL to identify the causative gene and generate tightly linked markers. One possible solution is to identify positional candidate genes by virtue of gene expression differences between MD resistant and susceptible chicken using deoxyribonucleic acid (DNA) microarrays followed by genetic mapping of the differentially-expressed genes. In this preliminary study, we show that DNA microarrays containing approximately 1200 genes or expressed sequence tags (ESTs) are able to reproducibly detect differences in gene expression between the inbred ADOL lines 63 (MD resistant) and 72 (MD susceptible) of uninfected and Marek's disease virus (MDV)-infected peripheral blood lymphocytes. Microarray data were validated by quantitative polymerase chain reaction (PCR) and found to be consistent with previous literature on gene induction or immune response. Integration of the microarrays with genetic mapping data was achieved with a sample of 15 genes. Twelve of these genes had mapped human orthologues. Seven genes were located on the chicken linkage map as predicted by the human-chicken comparative map, while two other genes defined a new conserved syntenic group. More importantly, one of the genes with differential expression is known to confer genetic resistance to MD while another gene is a prime positional candidate for a QTL.     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.     

Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease

Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte’s number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.     

A mitochondrial molecular marker of resistance to Bayoud disease in date palm

We have previously shown that two circular plasmid-like DNAs (the S and the R plasmids) can be found in the mitochondria of date palm (Phoenix dactylifera L.), a dioecious monocotyledonous tree. The two plasmids differ essentially by the absence, in the R plasmid, of a 109-bp DNA segment. Using 36 date palm varieties and employing a PCR-based approach, we show that the simultaneous presence of the R plasmid and absence of the S plasmid can be considered as a reliable molecular marker of resistance to a vascular wilt (Bayoud disease) caused by the fungus Fusarium oxysporum f. sp. albedinis. Conversely, the simultaneous presence of the S plasmid and absence of the R plasmid is correlated to Bayoud disease susceptibility The availibility of this diagnostic tool for plasmid characterization should subsequently allow simple, rapid and efficient selection of Bayoud-resistant individuals from the large number of date palms obtained by natural crosses which display good date quality. Received: 14 August 2000 / Accepted: 10 November 2000     

Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands.     

Major histocompatibility alleles associated with local resistance to malaria in a passerine

Malaria parasites are a major cause of human mortality in tropical countries and a potential threat for wildlife, as witnessed by the malaria-induced extinction of naive Hawaiian avifauna. Identifying resistance mechanisms is therefore crucial both for human health and wildlife conservation. Patterns of malaria resistance are known to be highly polygenic in both humans and mice, with marked contributions attributed to major histocompatibility (Mhc) genes. Here we show that specific Mhc variants are linked to both increased resistance and susceptibility to malaria infection in a wild passerine species, the house sparrow (Passer domesticus). In addition, links between host immunogenetics and resistance to malaria involved population-specific alleles, suggesting local adaptation in this host-parasite interaction. This is the first evidence for a population-specific genetic control of resistance to malaria in a wild species.     

Use of chicken microsatellite markers in turkey: a pessimistic view

Eighty-eight chicken microsatellite markers, previously developed in our laboratory, were tested for their ability to amplify polymorphic fragments using turkey genomic DNA. Amplification products were obtained for 61 chicken microsatellite markers (69.1%) whereas 27 (30.9%) did not give rise to any products, even when different polymerase chain reaction conditions were employed. From the 61 markers that gave a product, only eight showed a length polymorphism while 37 were monomorphic on the three divergent commercial turkey lines used. The remaining 16 markers yielded many unspecific bands and no specific amplification product could be obtained. Five polymorphic and eleven monomorphic products contained a detectable microsatellite repeat. Furthermore, of the markers that detected a polymorphism in turkey, the observed heterozygosity (15–50%) and allelic variation (only 2 in most cases) was very low. Therefore, on the basis of our results, we think that chicken microsatellite markers are not very useful for mapping purposes in turkey.     

Mitochondrial differentiation in a polymorphic land snail: evidence for Pleistocene survival within the boundaries of permafrost

The genetic differentiation of populations having colonized formerly unsuitable habitats after the Pleistocene glaciations depends to a great extent on the speed of expansion. Slow dispersers maintain their refugial diversity whereas fast dispersal leads to a reduction of diversity in the newly colonized areas. During the Pleistocene, almost the entire current range of the land snail Arianta arbustorum has repeatedly been covered with ice or been subjected to permafrost. Owing to the low potential for dispersal of land snails, slow (re)colonization of the wide range from southern refugia can be excluded. Alternatively, fast, passive dispersal from southern refugia or survival in and expansion from multiple refugia within the area subjected to permafrost may account for the current distribution. To distinguish between these scenarios we reconstructed a phylogeography based on the sequences of a fragment of the cytochrome oxidase I from 133 individuals collected at 45 localities and analysed the molecular variance. Seventy-five haplotypes were found that diverged on average at 7.52% of positions. This high degree of diversity suggests that A. arbustorum is an old species in which the population structure, isolation and the hermaphroditic nature have reduced the probability of lineage extinction. The genetic structure was highly significant with the highest variance partition found among regions. Geographic distance and mitochondrial differentiation were not congruent. Lineages had overlapping ranges. The clear genetic differentiation and the patchy pattern of haplotype distribution suggest that colonization of formerly unsuitable habitats was mainly achieved from multiple populations from within the permafrost area.