Phylogenetic Analysis of Field Isolates of Feline Calcivirus (FCV) in Japan by Sequencing Part of Its Capsid Gene

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B–F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.0%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH₂)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.     

Flow cytometry: clinical applications in equine medicine

The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell surface proteins. Other approaches have been used to elucidate changes in cell function and DNA content. This review is intended to provide the reader with the fundamental uses of flow cytometry. Examples of clinical applications in equine patients include immune-mediated hemolytic anemia, immune-mediated thrombocytopenia (IMT), chronic inflammatory disease, and neoplasia.     

Evaluation of the Abbott LCx Mycobacterium Tuberculosis Assay for Direct Detection of Mycobacterium Bovis in Bovine Tissue Samples

The commercial LCx amplification assay, usually employed to detect the Myocobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Löwenstein-Jensen solid medium and pathological findings on 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.     

DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.     

扁蓿豆遗传多样性研究初探

论述了扁蓿豆遗传多样性研究的依据,利用不同生境条件下生长的野生和栽培品种,采用形态标记、细胞标记、生化标记和分子标记的方法多方面地对扁蓿豆进行遗传多样性的研究。     

新扬州鸡早期增重的OPAY02-SCAR分子标记研究

OPAY02型2条多态性条带经克隆、测序和引物设计后,转换成SCAR标记,并对86个新扬州鸡随机交配后代基因组DNA进行了PCR扩增.2条带DNA序列与红色原鸡基因组序列比对结果表明,大分子量条带与位于红色原鸡第3号染色体上序列有98%的同源性,共检测到8个SNPS,其中195位的碱基T→G,316位的A→T,538位的G→A,731位的T→A,1 147位的G→A,1 329位的T→C,1 927位的C→T,2 081位的C→T,小分子量条带与红色原鸡没有同源序列,推测新扬州鸡野祖除红色原鸡外,还有其它来源.SCAR标记分析表明,经条件优化随机扩增的OPAY02型标记稳定、可靠,可用于遗传分析.2条带所在座位群体基因型平衡性测验结果表明,所测新扬州鸡群体处于平衡状态,选择可以打破平衡,有利于动物育种.     

牛基因组DNA两种提取方法的比较研究

研究哺乳动物基因组DNA的水煮抽提法，并将传统的酚仿抽提法和水煮法进行比较，水煮法即通过对细胞的煮沸和冷却，使细胞破裂、蛋白变性，从而获得用于PCR扩增的模板DNA的方法。通过电泳分析和PCR扩增检测了所提取基因组DNA的完整性、可行性。检测时采用3对引物对2种方法提取的DNA进行了PCR扩增，同时对冷冻保存的基因组DNA也进行了扩增检测。结果表明：水煮法提取基因组DNA是一种快速、简便、经济、高效的方法。