Evaluation of a new immunocapture test for the diagnosis of ovine brucellosis caused by Brucella melitensis

A new immunocapture technique has been applied to the diagnosis of ovine brucellosis under experimental conditions. The tests were made on a serum bank derived from both young and adult ewes vaccinated conjunctivally with the Rev 1 strain at a dose of 10(8) to 10(9) colony-forming units. Adult ewes were infected experimentally two-and-a-half years after they had been vaccinated and the results were compared with an unvaccinated control group. The condition of each animal in terms of infection with Brucella melitensis was determined by clinical and bacteriological investigations. The development of the immune response was compared by the rose bengal test, the complement fixation test, the Coombs' test and the immunocapture technique for 180 days after the vaccination and for 410 days after the experimental infection, that is, the two following gestations. The results suggest that the new technique is more specific in animals vaccinated conjunctivally, regardless of their age when they were vaccinated. After the experimental infection, significantly (P < 0.05) fewer of the vaccinated sheep which were free of clinical signs and were not excreting B melitensis reacted positively to the test.     

Early hepatic immune response in rats infected with Fasciola hepatica

We investigated the phenotype of the T cells (CD4+ and CD8+) that produced Th1 (IFN-gamma) and Th2 cytokines (IL-4 and IL-10) during the firsttwo weeks of experimental fasciolosis in rats. We also followed the kinetics of the cytokine and proliferative responses of hepatic mononuclear cells (HMNC) over the same period. We found that HMNC were more numerous in the infected animals than in the controls. The percentage of CD4+ cells increased significantly after infection, whereas the percentage of CD8+ cells did not change. Moreover, the frequency of the cells producing (CP) cytokine changed after infection. The frequency of CP IFN-gamma on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-gamma was clearly lower on day 14 pi, whereas the frequency of CP IL-4 and CP IL-10 had increased. The CP IL-10-were mostly CD4+. Mitogenic stimulation (phorbol myristate acetate/ionomycin) of HMNC led to an increase in the amounts of the Th2 cytokines in the supernatant on days 7 and 14 pi, with the increase more pronounced on day 14. In contrast, IFN-gamma levels also increased by day 7 pi but then decreased to below control levels by day 14. In addition, HMNC proliferation in response to mitogen followed a similar pattern to IFN-gamma production. These findings suggested that, during the first 2 weeks of infection, F hepatica induced a transient ThO cytokine profile followed by downregulation of the cellular response and the induction of a Th2 cytokine profile.     

Evaluation of the hepatic NK cell response during the early phase of Fasciola hepatica infection in rats

The in situ distribution of NK cells in rat liver during the first 28 days of an experimental infection with F hepatica was investigated. NK cells were distributed homogeneously throughout the hepatic parenchyma in uninfected animals. The total number of hepatic mononuclear cells increased significantly following infection, but the proportion of NK cells did not change. After infection, these cells were found around the portal space, around the centrolobular vein, in the periportal fibrosis and in the band of collagen. However, no NK cells could be detected in or around the granuloma during infection. The frequency of both I L-2- and IFNgamma-producing NK cells was higher on day 7 postinfection (pi) but only the percentage of IFNgamma -CD161+ subsets remained elevated thereafter, whereas the percentage of both IL-2+CD161+ and IL-4+CD161+ subsets returned to the baseline. The number of CD161+IL10+ cells did not change significantly. These results suggest that NK cells could be another source for the early production of IFNgamma but provide no evidence that these cells are involved in early events associated with granuloma formation.     

The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce

ABSTRACT Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo1(1) and mo1(2), are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo1(2) confers resistance with most isolates, the genes mo1(1) or mo1(2) confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo1(1), for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo1(2) lettuce lines because its systemic movement was restricted in mo1(2) lines but not in susceptible or in mo1(1) lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs.     

The afa-related gene cluster in necrotoxigenic and other Escherichia coli from animals belongs to the afa-8 variant

Six hundred and nine necrotoxigenic Escherichia coli type 1 and 2 (NTEC1 and NTEC2) and non-NTEC isolated in Western and Southern Europe, North Africa and Canada from diseased calves, pigs, humans, poultry, and 55 isolated from asymptomatic calves were studied for the identification of afa-related sequences to the recently described afa-7 and afa-8 gene cluster variants from two bovine Escherichia coli (Lalioui et al., 1999). Colony hybridization and PCR assays for the afaD-7, afaE-7, afaD-8 and afaE-8 identified the afa-related sequences to the afa-8 gene cluster in most (67/79; 85%) of the E. coli positive with the Afa-f family probe and in 14 additional strains negative with the Afa-f probe. No E. coli was positive for the afa-7 gene cluster. The existence of afa-8 positive strains was thus confirmed among bovine E. coli and for the first time among porcine, poultry and human E. coli. Sequencing of the afaE-8 amplicon of nine strains from the different host species showed a high degree of conservation (>95% at the DNA level; >92% at the amino-acid level). The afa-8 gene cluster was more frequent in E. coli from diseased calves (18%) than from piglets (12%), humans (6%) and poultry (5%). Bovine NTEC2 (26%) were more frequently positive than NTEC 1 (20%) and non-NTEC (11%). E. coli isolated from asymptomatic calves were rarely positive: one NTEC2 (3%) and no non-NTEC. The afa-8 gene cluster was located on the Vir plasmid in 11/23 NTEC2, but no plasmid localization was detected in NTEC1 or non-NTEC.     

A serological survey on classical swine fever (CSF), Aujeszky's disease (AD) and porcine reproductive and respiratory syndrome (PRRS) virus infections in French wild boars from 1991 to 1998

In early 1992, a CSF epizootic was clinically recognised in a wild boar population of approximately 1300 animals within an area of 250km(2) located in the east of France. In order to check the CSF situation in wild boars outside this area, a serological survey was carried out in the rest of France, for 8 consecutive years (1991-1998). This paper reports on the results obtained during this survey which included wild boars shot during the hunting period but also boars reared within fences. Around 1000-2700 sera a year were tested for the presence of antibodies to classical swine fever virus (CSFV) and also to Aujeszky's disease virus (ADV). Out of 12025 sera tested over the whole period, 80 wild boars were found positive for CSF antibodies. Sixty of them were collected on wild boars shot during the years 1992-1994 in the epizootic area located in east of France and 10 were collected in Corsica during the years 1994-1996. The last four positive samples were single reactors coming from areas or farms, which were thereafter confirmed to be serologically negative. These results together with the fact that no disease has been reported so far illustrate that the French wild boar population is probably not concerned by CSF infection (excepted in the east of France where the disease has now become enzootic). Two hundred and forty nine sera were initially detected as CSF positive but confirmed secondarily as positive for border disease (BD) antibodies. This finding shows that wild boars are also susceptible to infection by ruminant pestiviruses. Four hundred and twenty three wild boars have been found positive for ADV antibodies. In addition, from 1993 to 1995, 909 samples were tested for the presence of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV). Thirty three of them were positive. The results on AD and PRRS antibody detection show that wild boars may constitute a reservoir for various infectious diseases of pigs.     

Quantification of DNA of citrus huanglongbing pathogen in diseased leaves using competitive PCR

A quantification system for huanglongbing pathogen using a competitive polymerase chain reaction method and image-analyzing software were developed to obtain precise results. Significant differences in the quantity of pathogen were thus determined in leaves of two citrus cultivars commonly cultivated in southern Vietnam. Less pathogen-related DNA was detected from the tissue of citrus cultivars that are believed to be more tolerant than susceptible cultivars. The quantification system will be used in studies on pathogen proliferation and movement inside citrus tissue.     

灵芝发酵菌丝三萜类化合物含量的测定

建立了灵芝发酵菌丝三萜类化合物含量的检测方法。方法以齐墩果酸作为标准品．用香草醛一高氯酸发色体系进行比色．确定灵芝发酵菌丝三萜含量测定的实验条件．结果三萜类在20—140ug范围内有很好的线性关系．y=0．005x-0．019r=0．998。结论本法简便、准确、重复性好．可用于灵芝发酵菌丝三萜类含量的检测．以鉴定灵芝的品质及其产品的三萜含量。