Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.     

西安荷斯坦奶牛群5个基因座位遗传多态性的PCR-RFLP分析

应用PCR-RFLP方法对西安荷斯坦牛的κ-en、β-lg、β-lg5′侧翼区、CSN1S2、IGFBP-3共5个基因座位进行了多态性分析。结果表明，在西安荷斯坦牛群中，没有发现携带CSN1S2^P等位基因的个体，其多态信息含量为0。κ-en基因座位呈现低度多态(PIC=0．2366)，β-lg、β-lg5′侧翼区、IGFBP-3基因座位的多态信息含量分别为0．3168、0．3689、0．4439，均呈现中度多态。κ-en、β-lg、β-lg5′侧翼区、IGFBP-3、CSNIS2基因座位的杂合度和DNA多态度分别为0．2742、0．3947、0．4879、0．4891、0和0．0255、0．0116、0．0333、0．0112、0。而且，在西安荷斯坦牛群中，κ-en、β-lg、β-lg5′侧翼区、IGFBP-3共4个基因座位均处于Hardy-Weinberg平衡状态，CSN1S2基因座位处于纯合状态。     

亚洲璃眼蜱唾液腺一新功能基因的克隆与测序

HaB1是亚洲璃眼蜱雌成蜱唾液腺差异表达基因文库中的一个片段,根据其序列设计引物HaB1-GSP1和HaB1-GSP2,以唾液腺总RNA为模板,RACE法扩增获得HaB1的未知3′-末端。测定该末端序列,进行序列拼接,设计全长引物5-′CCAGTCCGAAGGAAGGGCG-3′和5-′TCTC-CGGGCACGTGAAGTGTC-3′。以cDNA第一链为模板,扩增获得基因全长。经核查表明,该基因为一新基因(登录号AY803896)。     

复方中草药"毒菌杀"对禽大肠杆菌及鸡白痢沙门氏菌的体外抑菌试验

观察了复方中草药“毒菌杀”的安全性及其对禽大肠杆菌及鸡白痢沙门氏菌的体外抑菌情况。结果发现“毒菌杀”安全性高，组成“毒菌杀”的各单味中药及其合剂对大肠杆菌的最低抑菌浓度MIC(g/mL)分别为板蓝根0．05、穿心莲0．05、黄芪0．025、黄柏0．05、柴胡0．05、生地0．05、甘草0．05、当归0．025，“毒菌杀”方剂为0．05；对沙门氏菌的MIC分别为板蓝根0．05、穿心莲0．025、黄芪0．025、黄柏0．05、柴胡0．025、生地0．05、甘草0．025、当归0．05，“毒菌杀”方剂为0．025；而牛胆汁对这两种致病菌的最低抑菌浓度分别为2％和1％。说明“毒菌杀”方剂对大肠杆菌、沙门氏菌具有明显的抑制作用。     

添加不同酵母培养物对瘤胃纤维分解菌群和纤维素酶活的影响

用3种酵母培养物(YC- 1、YC 2和YC- 3)分别饲喂4头带有永久性瘤胃瘘管的肉牛,研究培养物对瘤胃发酵、纤维分解酶活性和3种纤维分解菌数量的影响,结果表明:YC 2处理的乙酸、丙酸、丁酸和总 VFA浓度显著高于对照组(P<0 .05),YC 1和YC 3处理的乙酸/丙酸比例显著降低(P<0 .01);各处理均能显著提高瘤胃内羧甲基纤维素酶、水杨苷酶和木聚糖酶的活性(P<0 .01);各处理都显著提高黄化瘤胃球菌的相对比例(P<0 .01),16SrRNA特异性寡聚核苷酸探针杂交法分析测定结果表明 3 种纤维分解菌在瘤胃细菌中所占比例为 3. 80%±0 .2%。     

猪瘟病毒及其全长cDNA在猪肾细胞中增殖表达特性的比较研究

以猪瘟C-株弱毒疫苗株、经典强毒石门株和C-株全长cDNA为试验材料，以免疫荧光技术为主；结合ELISA和RT-PCR检测技术，对其在培养细胞中的增殖特性和表达规律进行了比较研究。同时筛选猪瘟病毒及其全长cDNA的敏感细胞，探索其增殖表达规律，以便为猪瘟病毒反向遗传操作提供技术方法和可操作的条件。     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH‐Cytochrome B5 Reductase Gene

Background

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Objectives

To analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.

Animals

Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.

Methods

Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.

Results

Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).

Conclusions and Clinical Importance

This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.