A Review of Methods Used in the Laboratory Diagnosis of Fireblight1)

Most well–known laboratory methods which can be used in the diagnosis of fireblight, as well as more recent developments in identification, have been discussed. They vary from the use of simple culture media to the use of immunofluorescent microscopy. The use of any one method alone is not advised, but by combining methods, accurate and rapid diagnosis is possible. In order to minimize diagnostic errors, especially among less experienced workers, the use of 5 % sucrose–nutrient agar for bacterial isolation followed by serological control is recommended. For further information reference is given particularly to the procedures described by Lelliott at the first EPPO Conference on Fireblight held in 1967 at Canterbury.     

我国兽用生物制品质量现状及改进措施

总结了近年来我国兽用生物制品质量监督抽检结果,对产品质量现状进行了分析,并就如何提高兽用生物制品质量提出了应对措施。     

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types.

LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR.

A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%).

DIAGNOSIS: Bovine haemoplasmosis.

CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.     

Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Effect of Dimethyl Sulfoxide on Cell Cycle Synchronization of Goldfish Caudal Fin Derived Fibroblasts Cells

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin‐derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

In‐field distribution of Plasmodiophora brassicae measured using quantitative real‐time PCR

A protocol using real‐time polymerase chain reaction (PCR) for the direct detection and quantification of Plasmodiophora brassicae in soil samples was developed and used on naturally and artificially infested soil samples containing different concentrations of P. brassicae. Species‐specific primers and a TaqMan fluorogenic probe were designed to amplify a small region of P. brassicae ribosomal DNA. Total genomic DNA was extracted and purified from soil samples using commercial kits. The amount of pathogen DNA was quantified using a standard curve generated by including reactions containing different amounts of a plasmid carrying the P. brassicae target sequence. The PCR assay was optimized to give high amplification efficiency and three to four copies of the target DNA sequence were detected. Regression analysis showed that the standard curve was linear over at least six orders of magnitude (R² > 0·99) and that the amplification efficiency was >92%. The detection limit in soil samples corresponded to 500 resting spores g^?1 soil. The intersample reproducibility was similar to, or higher than, that of assays for other pathogens quantified in soil samples. Bait plants were used to validate the real‐time PCR assay. The protocol developed was used to investigate the spatial distribution of P. brassicae DNA in different fields and a significant difference was found between in‐field sampling points. The reproducibility of soil sampling was evaluated and showed no significant differences for samples with low levels of inoculum, whereas at higher levels differences occurred. Indicator kriging was used for mapping the probability of detecting P. brassicae within a 2‐ha area of a field. A threshold level of 5 fg plasmid DNA g^?1 soil, corresponding to approximately 3 × 10³P. brassicae resting spores g^?1 soil, is suggested for growing resistant cultivars. The results provide a robust and reliable technique for predicting the risk of disease development and for assessing the distribution of disease within fields.     

Increasing expression of an anti-picloram single-chain variable fragment (ScFv) antibody and resistance to picloram in transgenic tobacco (Nicotiana tabacum)

Systematic research involving four chimeric gene constructions designed to express the same anti-picloram single-chain variable fragment (scFv) antibody is described. Agrobacterium-mediated transformation produced at least 25 transgenic tobacco plants with each of these, and the number of T-DNA loci in each plant was determined using kanamycin-resistance segregation assays. The relative amounts of active and total scFv in each plant were evaluated using quantitative enzyme-linked immunosorbent assay and immunoblot technologies, respectively. No significant differences in scFv activity were found among the four groups of single-locus plants, although the 35S/M construct was found to produce significantly more total anti-picloram scFv than the other three constructs. A dose-response bioassay involving T(1) seedlings from several of the highest expressers of active scFv demonstrated resistance to a constant exposure of picloram at 5 x 10(-)(8) M. Other approaches for increasing antibody-based herbicide resistance are discussed, as further improvements are needed before practical application of this technology.     

Seroprevalence of Canine Herpesvirus‐1 in the Belgian Dog Population in 2000

Canine herpesvirus‐1 (CHV‐1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme‐linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV‐1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV‐1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non‐breeding dogs.