Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines

The food grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.     

Auxin distribution in Lotus japonicus during root nodule development

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.     

Transglycosidase activity of chitotriosidase: improved enzymatic assay for the human macrophage chitinase

Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy.     

PCR for the detection of Streptobacillus moniliformis

Streptobacillus moniliformis is a Gram-negative bacterium found in various laboratory animal species and is the cause of rat bite fever and Haverhill fever in man. In order to evaluate a polymerase chain reaction (PCR) for the detection of this zoonotic bacterium in animal tissues a set of primers was designed based on the DNA base sequence of part of the 16S rRNA gene from 11 S. moniliformis strains. The PCR detected as few as 2-6 copies of S. moniliformis DNA. A 296 bp DNA fragment was amplified from S. moniliformis strains from rodents, humans and turkeys. Amplicons of about the same size were obtained from Fusobacterium necrogenes and Sebaldella (Bacteroides) termitidis but Bfa I treatment of these amplicons did not result in the S. moniliformis specific 130 bp DNA fragment. The in silico evaluation of 14 additional Fusobacterium spp. and 12 unculturable phytoplasmas indicated that none is likely to give rise to confusing amplicons or DNA fragments. The PCR detected S. moniliformis infection in all four orally- and four intravenously-infected C57BL/6 mice and the bacterium was cultured from all but one mouse. The PCR detected S. moniliformis infection in all 12 orally-infected WU rats, and in five of eight rats exposed to natural infection. Enzyme linked immunosorbent assay (ELISA) and PCR were equally successful in detecting infection in rats but S. moniliformis was not detected by using culture.     

Accumulation of lipochitin oligosaccharides and NodD-activating compounds in an efficient plant--Rhizobium nodulation assay

During legume plant--Rhizobium spp. interactions, leading to the formation of nitrogen-fixing root nodules, the two major determinants of host plant-specificity are plant-produced nod gene inducers (NodD protein activating compounds) and bacterial lipochitin oligosaccharides (LCOs or Nod factors). In a time course, we describe the accumulation of LCOs in an efficient nodulation assay with Vicia sativa subsp. nigra and Rhizobium leguminosarum, in connection with the presence of NodD-activating compounds in the exudate of V. sativa roots. Relatively small amounts of both LCOs and NodD-activating compounds were found to be required for initiation of nodulation during the first days after inoculation. A strong increase in the amount of NodRlv-V[18:4,Ac] LCOs preceded root infection and nodule primordium formation. In contrast to the situation with non-nodulating rhizobia and nonmitogenic LCOs, the amount of NodD-activating compounds in the culture medium remained small after addition of nodulating rhizobia or mitogenic LCOs. Furthermore, addition of nodulating rhizobia or mitogenic LCOs resulted in nearly complete inhibition of root hair formation and elongation, whereas nonmitogenic LCOs stimulated root hair growth. Retention of NodD-activating compounds in the root may inhibit root hair growth.     

Excretion of wild-type and vaccine-derived poliovirus in the feces of poliovirus receptor-transgenic mice

The emergence of circulating vaccine-derived poliovirus (cVDPV) strains in suboptimally vaccinated populations is a serious threat to the global poliovirus eradication. The genetic determinants for the transmissibility phenotype of polioviruses, and in particularly of cVDPV strains, are currently unknown. Here we describe the fecal excretion of wild-type poliovirus, oral polio vaccine, and cVDPV (Hispaniola) strains after intraperitoneal injection in poliovirus receptor-transgenic mice. Both the pattern and the level of fecal excretion of the cVDPV strains resemble those of wild-type poliovirus type 1. In contrast, very little poliovirus was present in the feces after oral polio vaccine administration. This mouse model will be helpful in elucidating the genetic determinants for the high fecal-oral transmission phenotype of cVDPV strains.     

Transmission of rat and guineapig Haemophilus spp. to mice and rats

Mice and rats, free from Pasteurellaceae, were exposed to Haemophilus spp. (V-factor dependent Pasteurellaceae) by housing in proximity to infected rats or guinea pigs, and monitored by culture and enzyme-linked immunosorbent assay (ELISA) for cross infection. A minority of mice became infected when exposed to Haemophilus-infected rats but none when exposed to guinea pigs. Rats were readily infected when exposed to Haemophilus-infected guinea pigs or rats. Although Pasteurellaceae infections are commonly considered as host specific, our data show that Haemophilus spp. can cross the species barrier from rats to mice and from guinea pigs to rats.     

Comparison of assays for antibodies to Encephalitozoon cuniculi in rabbits

Two indirect immunofluorescence (IIF) assays, two enzyme-linked immunosorbent assays (ELISAs) and the carbon immunoassay (CIA) for determination of antibodies to Encephalitozoon cuniculi were compared using 210 sera of rabbits, 135 of which originated from seven infected colonies, while 75 originated from four uninfected colonies. There was no evidence of a difference between the different assays with respect to the number of positive sera. There was a clear correlation between the quantitative response measured by IIF and CIA and the other assays, and between both IIF tests, while no such correlation was found in the quantitative response measured by ELISAs, which might be explained by the less quantitative nature of the ELISA. Therefore quantitative determination of antibodies to E. cuniculi should be performed by IIF and not by ELISA. The nosographic sensitivities N1 and specificities N2 of the assays were > or = 0.94 and > or = 0.97 respectively. Small differences in N1 and N2 between the assays, although not statistically significant, were responsible for differences in the calculated predictive values of a positive test and of a negative test. As expected, the magnitude of these differences depended on the fraction of positive sera sampled from a given colony. There was strong evidence of such a difference between the fraction of positive sera found in different colonies, but the sample size from some colonies was too small to allow any conclusion, whether this was due to differences in the prevalences of the infection in the colonies or something else. We conclude that any of the assays will be suitable for the routine health monitoring of laboratory rabbit colonies for E. cuniculi infection, as recommended by the Federation of European Laboratory Animal Science Associations.