Genomic organization of the T-cell receptor gamma gene and PCR detection of its clonal rearrangement in canine T-cell lymphoma/leukemia

Because the T-cell receptor gamma (TCRgamma) gene is rearranged at an early stage of T-cell development in both TCRalphabeta and TCRgammadelta lineages, it has been preferentially targeted to detect T-cell clonality in human lymphoma/leukemia. We isolated 22 independent cDNA clones encoding canine TCRgamma and the following analysis of nucleotide sequences using the dog genome database revealed that the canine TCRgamma locus contains at least four repertories of variable genes that can be organized into two distinct subgroups and six repertories of joining genes belonging to two distinct subgroups according to the nucleotide sequence similarity. The findings allowed us to design PCR primers that were directed to the conserved or specific nucleotide sequences for each subgroup of variable and joining genes. By using four different combinations of primers, a PCR-based analysis was performed on cell samples collected from T-cell lymphoma/leukemia and B-cell lymphoma cases and hyperplastic and normal lymph nodes. All cell samples from 11 T-cell malignancy cases exhibited clonal amplification by two out of four primer combinations. This finding was considered to be valuable in PCR-based analysis for detecting T-cell clonality in canine lymphoma/leukemia.     

核酸水平检测犬副流感病毒方法的建立

根据GenBank中与犬副流感(CPIV)同源性较近的SV5病毒N基因保守序列,利用DNAStar软件设计了一对特异性引物,能扩增265bp大小的片段,并以此建立了检测CPIV的RT-PCR方法,实验证明该对引物特异扩增CPIV;不扩增犬瘟热病毒、犬细小病毒、犬腺病毒和狂犬病病毒犬的四种病原的核酸。检测临床病料20份,其中2分为CPIV阳性。此法敏感性较高。是检测犬急性传染性呼吸道疾病(CIRD)中CPIV的有效的方法。     

GeneScanning analysis of Ig/TCR gene rearrangements to detect clonality in canine lymphomas

The diagnosis of canine lymphoma is achieved using morphological and immunological methods. In a certain percentage of cases, difficulties in making a definitive diagnosis of lymphoproliferative disorders may occur despite extensive immunophenotyping. Therefore, additional diagnostics, such as molecular assessment of Ig/TCR gene rearrangements clonality, may confirm the final diagnosis. Polyacrylamide gel electrophoresis and heteroduplex analysis have already been proven to be suitable for detecting clonality but are cumbersome and labor-intensive. In the present study, GeneScanning analysis of PCR products originating from different primer sets targeting different regions of Ig and TCR was validated in improving sensitivity as well as in reducing the turnaround time of gene rearrangement assays. GeneScanning exploits 5' fluorescently labelled primers for the automated and fast analysis of PCR products either as singleplex or multiplex runs. Initially, the assay was set up using DNA purified from normal tissues (n=6), hyperplastic/reactive tissues (n=10) and a small set of immunophenotyped lymphoma samples (n=12). The optimized methods were then used in a large set of 96 canine lymphoma samples. Normal and hyperplastic/reactive lymphoid tissues showed typically polyclonal or, occasionally, oligoclonal PCR products. Lymphoma samples showed monoclonal peaks arranged as a single or, occasionally, a double narrow base peak sometimes embedded in a polyclonal background. In all immunophenotyped cases, an Ig or TCR clonal finding corresponded to B- and T-cell lymphomas, respectively. Overall, 94/96 (97.9%) samples showed clonal Ig/TCR clonal rearrangements among which clonal Ig was found in 61/96 (63.5%) of samples and clonal TCR in 33/35 Ig negative samples (34.4% of all cases). In one out of ten randomly chosen cases, both Ig and TCR clonal gene rearrangements were found. Among the factors affecting assay accuracy, DNA quality has been shown to be critical and the amplification of DNA controls of different size are recommended to evaluate DNA integrity. Frozen material such as that which remained inside the hub of the needle used for diagnostic procedures is optimal for the analysis herein described. In conclusion, GeneScanning represents a versatile tool for routinely assessing Ig/TCR clonal rearrangements and supporting the diagnostic protocol of canine lymphomas.     

Rearrangement patterns of the canine TCRγ locus in a distinct group of T cell lymphomas

The T cell antigen receptor chains are assembled through a rearrangement process that combines variable (V), diversity (D) and joining (J) region genes. Recently, the entire canine T cell receptor γ (TRG) locus was described. It is arranged in 8 cassettes with up to 3 V genes, 2 J genes and 1 C gene each. However, no data is available beyond the level of sequence analysis. The objective of this study was to identify rearranged genes of the canine TRG locus through experimental analysis and to assess gene usage and patterns of rearrangement in a series of canine T cell lymphomas. Rearranged genes were identified through computational analysis of recombination signal sequences (RSSs), a gene's potential to generate a polyclonal smear, and through sequencing of clonal rearrangements in a series of T cell lymphomas. Out of a total of 32 Vγ and Jγ genes, 21 genes were found to rearrange, 8 genes were considered not rearranged and 3 genes were suspected to rearrange but their status could not be determined definitely. Rearrangements of the canine TRG locus were assessed in a group of canine T cell lymphomas as well as 3 neoplastic T cell lines. An average of 4.6 rearrangements per lymphoma was found suggesting that canine T cells routinely rearrange multiple cassettes per allele. The most commonly rearranged Vγ genes belonged to subgroups Vγ2, Vγ3, and Vγ7. Genes in cassettes 2 and 3 preferentially rearranged within their respective cassettes, while Vγ genes in cassette 7 rearranged to a Jγ gene in cassette 8. There was a strong preference for Vγ2 genes to rearrange to a 3' Jγ gene and for Vγ3 and Vγ7 genes to rearrange to a 5' Jγ gene. This rearrangement pattern coincided with the conservation of the spacer sequence between V and J gene subgroups rather than the topologic location of genes. These data show that highly divergent spacer sequences allow for equally efficient recombination and suggest that spacer sequences can mediate compatibility between V and J genes.     

FC‐14 Clonality studies of feline cutaneous lymphocytosis

A previous study described cutaneous lymphocytosis (CL) in 23 cats. The process resembles cutaneous pseudolymphoma in humans, a heterogeneous group of benign reactive proliferations of well‐differentiated lymphocytes in the skin of humans. Morphological and immunophenotypic characteristics do not offer reliable criteria to accurately predict the clinical outcome of feline CL or pseudolymphoma in humans. Presence of clonal cell populations is more consistent with a neoplastic process. In a previous study, feline CL lesions (20 cats) were evaluated for clonality using PCR, and only two cats had monoclonal T‐cell populations. Because false‐negative results may occur, the purpose of this study was to repeat the PCR using a revised primer set based on analysis of additional feline T‐cell receptor γ (TCRγ) sequences. DNA was isolated from 29 skin lesions and six internal organs of 20 cats. DNA integrity was assessed by glyceraldehyde‐3‐phosphate dehydrogenase PCR. Polymerase chain reaction clonality was performed using the revised primer set specific for feline TCRγ, and duplicate samples were evaluated. The PCR products were assessed by heteroduplex analysis. Clonal rearrangement of TCRγ was detected in 14 cats (24 of 35 tissues: 21 of 29 skin lesions and three of six internal organs); eight of these cats are still alive and six were euthanized. Monoclonal populations were seen in three of five cats that had involvement of internal organs. These findings indicate that feline CL is best considered as a slowly progressive process which may be reactive, but often evolves into a low‐grade indolent lymphoma. Funding: George H. Muller Fund for Research in Dermatology.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

7种猪场常见高致死病原GeXP检测方法的建立

本研究旨在建立能同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV2)、坏死梭杆菌(Fn)和副猪嗜血杆菌(Hps)7种猪场常见高致死性流行病原的多重PCR检测方法。利用7种病原体的保守序列设计7对特异性引物,同时合成了Cy-5标记的通用引物。将通用引物分别连接到特异性引物的5′端形成7对特异性嵌合引物。优化反应条件,分别使用7组嵌合引物和通用引物混合,扩增7种病原的混合cDNA/DNA,验证其单重PCR的特异性。利用GeXP多重基因表达遗传分析系统,混合7种嵌合引物和通用引物,扩增单一病原的cDNA/DNA,验证其多重PCR特异性;将其他常见猪病病原的基因组作为干扰的阳性标本,利用7对混合嵌合引物和通用引物进行多重PCR分析,扩增加入了阳性标本的混合模板,验证其多重PCR的抗干扰能力。利用重组质粒和体外转录的RNA进行梯度稀释,确定GeXP多重检测体系的灵敏度。结果表明,7种不同引物分别进行GeXP单重及多重检测,均能检测出特异性目的片段的信号,无明显的干扰片段信号出现;GeXP多重检测抗干扰试验结果显示,在混入3种干扰病原模板后,依然可同时特异性检测出7种病原;GeXP多重检测灵敏度分析显示,在10~3拷贝/μL浓度条件下能检测到7种不同基因的特异性结果。本研究建立的同时检测7种猪场常见高致死性流行病原的GeXP检测方法具有高通量、高特异性和高灵敏度的特点,为快速诊断猪流行性疾病的交叉感染和混合感染提供了新型的检测方法。     

T lymphocyte subpopulations diverge in commercially raised chickens

To evaluate immunocompetence in commercially raised chickens, we immunophenotyped Dekalb Delta and H&N White Leghorn (WLH) hybrids, 20 chickens in each of 3 age groups (9 wk [juvenile], 25 wk [young adult], and 79 or 80 wk [adult]), for circulating CD3+, CD4+, CD8+, TCR1+, TCR2+, and TCR3+ lymphocytes. The proportion of CD3+ T cells, including CD4+ and CD8+ subsets, was increased in the hybrids as compared with published values for laboratory-raised outbred WLH chickens. The proportion of the TCR2+ (Vbeta1) T cell subpopulation was also increased. An age-related decrease in the proportion of TCR1+ (gammasigma) T cells was noted in both hybrids. Further, a remarkably low CD4:CD8 ratio was evident in all age groups of both hybrids, indicating decreased immunocompetence. Overall, these experiments provide age-related proportions of various peripheral-blood T lymphocyte subpopulations in commercially raised Dekalb Delta and H&N chickens that diverge from the proportions in laboratory-raised outbred WLH chickens and suggest reduced immunocompetence. Such a decline in immunocompetence, including humoral immune capacity, could be attributed to genetic selection for production traits, environmental factors associated with commercial operations, and intense immunization.     

禽白血病病毒B、E和J亚群基因芯片检测方法的建立

目的基于多重PCR技术,建立禽白血病病毒(ALV)的B、E、J亚群的基因芯片分型和检测方法。方法根据NCBI已收录的ALV三个亚群的参考毒株cDNA序列,在各亚群特异性基因突变区两端选取其保守区域,设计合成三个亚群的通用上游引物1条,以及B、E亚群的通用下游引物和J亚群下游引物各1条,将上述引物用Cy3标记,建立多重PCR体系;参考靶序列内部的三个亚群各自的保守区域,选择亚群之间基因突变位点多的区域,设计合成5条寡核苷酸探针,制作寡核苷酸探针基因检测芯片;以寄主细胞DF-1中提取传代ALV的cDNA,以及合成NCBI收录的各亚群参考毒株的cDNA序列作为检测模板;利用Cy3标记的PCR扩增产物,与基因芯片进行杂交反应,扫描结果。结果芯片准确检测并分型三个亚群的参考毒株,其检测灵敏度能够达到102个基因拷贝,且与禽类常见的四种病毒均无交叉反应。结论本研究结果证明,基因芯片技术是一种ALV的B、E和J亚群进行检测和分型的有效方法,且具有较高的特异性和灵敏度,为今后在临床应用中快速鉴别诊断ALV等免疫抑制病提供可行性。     

鹅细小病毒和番鸭细小病毒双重PCR检测方法的建立

根据GenBank上登录的鹅细小病毒(GPV)和番鸭细小病毒(MDPV)基因序列,分别设计合成针对GPV非结构蛋白(NS)和MDPV NS2-VP1基因片段的2对引物GPV U/L和MDPV U/L,将GPV和MDPV提取核酸混合后作为模板,优化PCR反应条件,建立了能同时检测这2种病毒的双重PCR。特异性试验结果显示,引物GPV U/L仅特异性扩增出GPV-GZ1和GPV-GZ2株730bp核酸片段,引物MDPVU/L仅特异性扩增出MDPV的624bp核酸片段,双重PCR扩增出长度分别为730bp和624bp的2条特异性片段,而扩增鸭瘟病毒(DPV)和鹅副黏病毒(GPMV)的核酸扩增结果均为阴性。敏感性试验结果显示,双重PCR能同时检测到14.4pg的GPV核酸和28.8pg的MDPV核酸。结果表明,建立的双重PCR可用于GPV和MDPV的鉴别诊断和联合检测。     

Infrequent detection of papillomaviral DNA within canine cutaneous squamous cell carcinomas, haemangiosarcomas and healthy skin on the ventrum of dogs

Background – Canine squamous cell carcinomas (SCCs) most frequently develop on the ventral abdomen and are thought to be caused by ultraviolet (UV) light. Papillomaviruses (PVs) have been associated with cutaneous SCCs in multiple species, including dogs. Hypothesis – That PVs act as cofactors in canine UV‐induced SCCs. Animals – The study was performed on skin from the ventrum of 60 dogs. These samples included 20 SCCs, 20 haemangiosarcomas and 20 samples of clinically normal skin. Two canine viral plaques were included as positive controls for PV. Methods – PCR was used to amplify PV DNA from all samples. Primers used included two sets of consensus primers and two sets of primers that were designed specifically to amplify PV DNA sequences detected in the viral plaques. Results – The MY09/11 consensus primers amplified PV DNA from both viral plaques. One plaque contained a DNA sequence (CfPV‐JM) that had been previously reported from a dog with multiple cutaneous SCCs. The other plaque contained a previously unreported PV DNA sequence. No PV DNA was amplified by either consensus primer from any of the ventrum skin samples. Primers designed specifically to amplify the CfPV‐JM sequence amplified DNA from one SCC, but no other sample. No PV DNA was amplified using the other specific PCR primer set. Conclusions and clinical importance – These results do not support a significant role for PVs in SCC development from the ventrum of dogs. However, they contribute another PV sequence to the list of PVs that have been associated with viral plaque development in dogs.     

基于Nc2和Nc5基因的新孢子虫PCR方法的建立

本试验筛选了新孢子虫病PCR检测的引物,运用《新孢子虫病检疫技术规范》(SN/T 3499-2013)对根据犬新孢子虫Nc2和Nc5基因设计的PCR引物进行了评价。此外,同时运用F1/R1、F2/R2和SN/T 3499 F/SN/T 3499 R共同对14份荷斯坦牛和19份西门塔尔牛全血DNA进行PCR检测,旨在筛选出特异性较好的引物,建立新孢子虫病PCR检测方法和了解当地不同品系牛患新孢子虫病的感染率。结果显示,3对引物分别扩增出105、128和231 bp目的片段,均与预期目的片段大小相符;其中,F1/R1与SN/T 3499 F/SN/T 3499 R的最低检测量相同,为19.9 fg/μL,F2/R2最低检测量为199 fg/μL,说明F1/R1和SN/T 3499 F/SN/T 3499 R引物的敏感性更好;运用F1/R1、F2/R2引物分别对19.9 pg/μL和199 fg/μL模板重复进行4次扩增,均出现了较明亮的扩增条带,证明两对引物重复性较好。33份血液样品共检出6份阳性DNA,阳性率分别为21.43%和15.79%,检出复合率为100%。以上结果说明F1/R1和F2/R2引物均可作为新孢子虫病PCR的诊断引物,本试验初步建立了新孢子虫PCR方法,同时初步了解了当地牛群中新孢子虫感染情况,为有效预防和控制新孢子虫病提供了科学的理论依据。     

Establishing the Screening PCR Diagnostic Method of Neosporacaninum Based on Nc2 and Nc5 Genes

To screen applicable PCR diagnostic primers of Neosporacaninum based on Nc2 and Nc5 genes,two pairs of primer were designed according to the Nc2 and Nc5 genes sequences conserved region and"Quarantine Protocol For Neosporosis"(SN/T 3499-2013)were applied to verify the characteristic of primers.14 Holstein and 19 Simmental cattles blood DNA were detected using these primers by PCR to select specific primers which would be used to establish the new detection method and understand the neosporiasis infection rate of different local cattle farms.The results showed that 105,128 and 231 bp gene fragments were amplified using three pairs of primers,which were consistent with the expected fragment size.The F1/R1 and SN/T 3499 F/SN/T 3499 R minimum detectable amount both were 19.9 fg/ μL and the minimum amount of F2/R2 was 199 fg/ μL,indicating that F1/R1 and SN/T 3499 F/SN/T 3499 R were more sensitive than F2/R2.The bands were bright when used F1/R1,F2/R2 primers to amplify 19.9 pg/μL and 199 fg/μL DNA samples,proving two primers had good repeatability.6 positive samples were detected among 33 blood samples,and the positive rate was 21.43% and 15.79%,the recombination rate was 100%.It showed that F1/R1 and F2/R2 primer were both suitable to detect eneosporosis by PCR and the method was preliminary established.The condition of local cattle Neosporacaninum infections were learned.This study could provide a scientific basis for the effective prevention and control of neosporosis.     

莱航鸡rDNA重复基因家族的克隆

动物rDNA基因是一种GC含量较高、结构复杂的重复序列。通过结合生物信息学技术,经反复摸索后选用LAPCR法扩增莱航鸡rDNA基因重复序列,经测序鉴定最终克隆了莱航鸡的3个rDNA基因及其2个间隔序列。研究对克隆复杂DNA序列时引物设计的特别规则、反应体系的改进、DNA聚合酶的选用、循环参数的调整等进行了探索。     

犬源牛犬细小病毒和犬圆环病毒二联PCR检测方法的建立及应用

本研究旨在建立可同时检测犬源牛犬细小病毒(CBoV)和犬圆环病毒(CCV)的二联PCR检测方法,并对两种病毒病当前的流行情况进行监测和调查.分别将已发表的CBoV和CCV基因组序列进行同源性比对,选择高同源区段,应用Primer Primier 5计算机软件设计并合成了2对特异性扩增引物,目的片段大小分别为170 bp...     

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.     

兔巴氏杆菌和波氏杆菌多重PCR检测方法的建立

参考GenBank中兔巴氏杆菌16SrRNA和波氏杆菌的fim2的基因序列,应用Premier 5.0软件在二者高度保守区设计了2对引物,建立了适合巴氏杆菌和波氏杆菌快速检测的多重PCR检测方法。以该方法对巴氏杆菌和波氏杆菌参考菌株进行PCR扩增,分别能从各自的基因组中扩增出与试验设计相符的644bp和425bp的特异性DNA片段。将扩增所得的DNA片段进行克隆测序,测序结果表明分别为巴氏杆菌16SrRNA和波氏杆菌fim2基因序列。该方法对波氏杆菌的检测下限为4×102 CFU,对巴氏杆菌的检测下限为6×101 CFU。对兔源性沙门菌、葡萄球菌、大肠杆菌、魏氏梭菌扩增结果为阴性。表明所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于鉴别诊断兔巴氏杆菌和波氏杆菌以及2者混合感染。     

Detection of Theileria ovis in naturally infected sheep by nested PCR

A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 10(7) sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep.