IDT Biologika (Germany)

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

The small Indian mongoose (Urva aurpunctata) is a non-indigenous wildlife reservoir for rabies virus on several Caribbean islands. Oral rabies vaccination (ORV) has been suggested to prevent the spread of rabies in mongooses, but there are limited data on ORV bait survival in maritime/tropical climates. We compared disappearance rates of an egg-flavored bait block vs. a control (canned tuna) and for bait blocks, we assessed if season, habitat, and day/night would influence the disappearance rate. On the island of St. Kitts, West Indies, we baited 45 stations (n=35 bait blocks, n=10 controls) at dawn and checked them twice daily for 4 consecutive days. This procedure was repeated after a two-day period, for each combination of season (dry and wet) and habitat type (mixed grassland, littoral and dry forest). We used the Kaplan-Meier method to estimate bait survival at 12 and 36 hours after baiting. Across all sites, control baits were removed faster than bait blocks, but by 36 hours, over 90% of all baits had disappeared. For bait blocks only, the disappearance rate was higher during the dry season and in the dry forest habitat compared to the other habitats. There was no difference between diurnal and nocturnal disappearance rates. While the exact species that consumed the baits remain uncertain, we found that mongooses readily remove bait blocks, along with non-native mammals (e.g., dogs and cats) and land crabs in littoral habitats. We suggest that the bait blocks used in this study may be suitable for use as an external bait matrix for ORV baits.

To evaluate the long-term immunogenicity of the live-attenuated, oral rabies vaccine SPBN GASGAS in a full good clinical practice (GCP) compliant study, forty-six (46) healthy, seronegative red foxes (Vulpes vulpes) were allocated to two treatment groups: group 1 (n = 31) received a vaccine bait containing 1.7 ml of the vaccine of minimum potency (106.6 FFU/mL) and group 2 (n = 15) received a placebo-bait. In total, 29 animals of group 1 and 14 animals of group 2 were challenged at 12 months post-vaccination with a fox rabies virus isolate (103.0 MICLD50/mL). While 90% of the animals offered a vaccine bait resisted the challenge, only one animal (7%) of the controls survived. All animals that had seroconverted following vaccination survived the challenge infection at 12 months post-vaccination. Rabies specific antibodies could be detected as early as 14 days post-vaccination. Based on the kinetics of the antibody response to SPBN GASGAS as measured in ELISA and RFFIT, the animals maintained stable antibody titres during the 12-month pre-challenge observation period at a high level. The results indicate that successful vaccination using the oral route with this new rabies virus vaccine strain confers long-term duration of immunity beyond one year, meeting the same requirements as for licensure as laid down by the European Pharmacopoeia.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Background: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development.

BackgroundOral vaccination of the small Indian mongoose against rabies has been suggested as a potential tool to eliminate mongoose-mediated rabies on several Caribbean islands. A recently developed oral rabies virus vaccine strain, SPBN GASGAS, has already been shown to be efficacious in this reservoir species. Since, all available oral rabies vaccines are based on replication-competent viruses and vaccine baits are distributed unsupervised in the environment, enhanced safety standards for such vaccine types are required.ResultsThe results of safety studies, including overdose, repeated doses, dissemination and different routes of administration, in the target species are presented. It was shown that the construct was apathogenic, irrespective of dose and route of administration. Even when it was inoculated directly in the brain, it did not induce rabies infection. Furthermore, the vaccine strain did not spread within the target species after direct oral instillation beyond the site of entry.ConclusionThe vaccine strain SPBN GASGAS meets the safety requirements for live rabies virus vaccines in this target species, the small Indian mongoose.

BackgroundA diversifying pool of mammalian‐adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood.ObjectivesEfficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost‐effective large‐scale analysis.MethodsNew SIV haemagglutinin (HA) and neuraminidase (NA) subtype‐ and lineage‐specific multiplex real‐time RT‐PCRs (RT‐qPCR) have been developed and validated with reference virus isolates and clinical samples.ResultsA diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M‐gene‐specific influenza A virus RT‐qPCR. In a second step, positive samples are examined by tetraplex HA‐ and triplex NA‐specific RT‐qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages “av” (European avian‐derived), “hu” (European human‐derived) and “pdm” (human pandemic A/H1N1, 2009) are distinguished by RT‐qPCRs, and within the NA subtype N1, lineage “pdm” is differentiated. An RT‐PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT‐qPCR subtyping.ConclusionsThese new multiplex RT‐qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

Inactivated whole-virus vaccines against porcine parvovirus (PPV) can prevent disease but not infection and virus shedding after heterologous virus challenge. Here, we showed that the same is true for a homologous challenge. Pregnant sows were vaccinated with an experimental inactivated vaccine based on PPV strain 27a. They were challenged on day 40 of gestation with the virulent porcine parvovirus PPV-27a from which the vaccine was prepared (homologous challenge). On day 90 of gestation, the fetuses from vaccinated sows were protected against disease, while the fetuses of the non-vaccinated sows (control group) exhibited signs of parvovirus disease. All gilts, whether vaccinated or not vaccinated, showed a boost of PPV-specific antibodies indicative of virus infection and replication. Low DNA copy numbers, but not infectious virus, could be demonstrated in nasal or rectal swabs of immunized sows, but high copy numbers of challenge virus DNA as well as infectious virus could both be demonstrated in non-vaccinated sows.