Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle

BACKGROUND: DNA vaccination has been shown to elicit specific cellular and humoral immune responses to many different agents in a broad variety of species. However, looking at a commercial use, the duration of the immune response against the vaccine is critical. Therefore the persistence of the DNA vaccine, as well as its expression, should be investigated. We conducted these investigations on a DNA vaccine against Chlamydophila psittaci, a Gram-negative intracellular bacterium which causes respiratory disease in turkeys and humans. Previous studies showed that the DNA vaccine confers partial protection against C. psittaci infection in turkeys. Turkeys were injected intramuscularly with the DNA vaccine : a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer membrane protein (MOMP) of an avian C. psittaci serovar D strain. Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR) and mRNA translation by immunofluorescence staining of muscle biopsies. RESULTS: The results indicate that the DNA vaccine persists in turkey muscle for at least 10 weeks. Moreover, during this period of time MOMP was continuously expressed, as evidenced by the immunofluorescence staining and RT-PCR. CONCLUSION: Since C. psittaci infections occur at the age of 3 to 6 and 8 to 12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore, further research should concentrate on improving the elicited immune response, more specifically the cell-mediated immune response, rather than prolonging the lifespan of the plasmid

Kasveissa tuotettu rokote porsaiden vieroitusripuliin

Enterotoksiset Escherichia coli-kannat (ETEC) ovat yleisiä porsas- ja sikaripulin aiheuttajia maailmanlaajuisesti. Vieroitusvaiheessa (3-5 viikon iässä) porsaiden vastustuskyky on heikoimmillaan, koska imetyksen mukana ternimaidosta saadut vasta-aineet vähenevät ja porsaiden oma vasta-aineiden tuotto on vasta alkamassa. Tällöin haitalliset suolistopatogeenit (bakteerit tai virukset) pääsevät helposti kolonisoimaan ohutsuolen ja aiheuttamaan ripulin. Ripuli aiheuttaa paitsi turhaa kärsimystä porsaille, myös tuotannon heikkenemistä kasvun hidastuessa ja ääritapauksissa porsaskuolemia. Hygienian, kasvatusolosuhteiden ja ravinnon korkea taso ovat ensisijaisen tärkeitä pyrittäessä ehkäisemään ripulisairauksien puhkeamista, mutta usein joudutaan myös turvautumaan yksittäisten eläinten rokottamiseen tai antibiootteihin. Antibioottien käyttöä pyritään nykyään yleisesti rajoittamaan, sillä niiden leviäminen ympäristöön johtaa väistämättä antibiooteille vastustuskykyisten bakteerikantojen kehittymiseen. Vieroitusripulin ehkäisemiseksi kaivataankin uusia, sekä inhimillisesti, taloudellisesti että ajankäytöllisesti parempia keinoja. Kasvibiotekniikka mahdollistaa porsaiden ripuliongelman ehkäisemisen uudella tavalla. Rehukasveissa voidaan tuottaa ns. syötäviä kasvirokotteita, jotka aktivoivat elimistön oman puolustusjärjestelmän (immuunivasteen), minkä seurauksena eläin välttyy tietyn patogeenin aiheuttamalta taudilta. Tässä työssä olemme siirtogeenitekniikan avulla tuottaneet sinimailasessa ETEC bakteerin F4 fimbrian tartuntaproteiinia (FaeG). Tämä rokoteproteiini kohdennettiin viherhiukkasiin, jolloin siirtogeeniset sinimailaset tuottivat sitä 1% liukoisesta proteiinistaan. Kasvissa tuotettu rokoteproteiini todettiin kestäväksi ja se säilyi muuttumattomana kuivatussa sinimailasmateriaalissa ainakin kahden vuoden ajan. Porsaskokeessa vierotetuille porsaille annettiin rokoteproteiinia sisältävää sinimailasta. Kasvissa tuotettu rokoteproteiini sai porsaissa aikaan heikon F4-spesifisen seerumin vastaainereaktion, jota pystyttiin vahvistamaan, kun rokoteproteiinin tehostajana käytettiin koleratoksiinia. Kun koeporsaat altistettiin patogeenisellä F4+ ETEC-kannalla, koleratoksiinilla tehostettu rokoteproteiini pystyi myös vähentämään ulosteessa erittyvien ETEC-bakteerien määrää merkittävästi verrattuna kontrolliryhmään, joka oli käsitelty ei-siirtogeenisellä sinimailasella. Tulokset osoittavat, että kasvissa tuotettu FaeG rokoteproteiini käynnistää porsaissa F4-vasta-ainetuotannon ja vähentää F4+ETEC-infektion vakavuutta

Bioluminescent Imaging of Trypanosoma brucei Shows Preferential Testis Dissemination Which May Hamper Drug Efficacy in Sleeping Sickness

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs

Maternal colostral leukocytes appear to enhance cell-mediated recall response, but inhibit humoral recall response in prime-boost vaccinated calves

Whether colostral leukocytes (CLs) of vaccinated dams influence the immune response of neonatal calves following vaccination against the same antigen as their respective dams remains unanswered. Therefore, we compared the induction of humoral and cellular immune responses after vaccination in calves that had received CL-free or maternal CL-enriched colostrum from a cell-free colostrum bank of nonvaccinated cows. Also, vaccinated calves that had received fresh maternal colostrum from their own dam were included in the study. Moreover, we analyzed whether the post-partum time of priming vaccination (day 2, 5 or 10) of the calves could influence the outcome of the immune responses. All calves received a booster vaccination 23 days after the priming vaccination. All calves showed only an increase in tetanus toxoid (TT)-specific antibodies and TT-induced proliferation after booster vaccination. Tetanus toxoid-specific antibody responses in calves increased immediately after booster vaccination, irrespective of whether or not their cell-free bank colostrum had been enriched with CLs from their own dam. Conversely, calves receiving their own plain dam colostrum displayed a later humoral response, due to colostral antibodies. After booster vaccination, calves of the CL-enriched colostrum group had a more pronounced antigen-specific proliferative response than the calves of the CL-free colostrum group. We propose that CLs might have a suppressive influence on the emergence of the TT-specific antibodies, but an enhancing effect on the TT-specific lymphocyte proliferation of newborn calves upon TT vaccination, which is dependent on the time point of the priming vaccination.publisher: Elsevier articletitle: Maternal colostral leukocytes appear to enhance cell-mediated recall response, but inhibit humoral recall response in prime–boost vaccinated calves journaltitle: Journal of Reproductive Immunology articlelink: http://dx.doi.org/10.1016/j.jri.2015.11.004 content_type: article copyright: Copyright © 2016 Published by Elsevier Ireland Ltd.status: publishe

New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels.

BACKGROUND:Trypanosoma (T.) evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia. METHODOLOGY/PRINCIPAL FINDINGS:T. evansi was isolated in mice by inoculation with the cryopreserved buffy coat of parasitologically confirmed animals. Fourteen stocks were thus isolated and subject to genotyping with PCRs targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, minisatellite markers and the F1-ATP synthase γ subunit gene. Nine stocks corresponded to type A, two stocks were type B and three stocks represented mixed infections between A and B, but not hybrids. One T. evansi type A stock was completely akinetoplastic. Five stocks were adapted to in vitro culture and subjected to a drug sensitivity assay with melarsomine dihydrochloride, diminazene diaceturate, isometamidium chloride and suramin. In vitro adaptation induced some loss of kinetoplasts within 60 days. No correlation between drug sensitivity and absence of the kinetoplast was observed. Sequencing the full coding sequence of the F1-ATP synthase γ subunit revealed new type-specific single nucleotide polymorphisms and deletions. CONCLUSIONS/SIGNIFICANCE:This study addresses some limitations of current molecular markers for T. evansi genotyping. Polymorphism within the F1-ATP synthase γ subunit gene may provide new markers to identify the T. evansi type that do not rely on variant surface glycoprotein genes or kinetoplast DNA

New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels

Trypanosoma (T.) evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia.status: publishe

Exacerbation of Chlamydophila psittaci pathogenicity in turkeys superinfected by Escherichia coli

Both Chlamydophila psittaci and Escherichia coli infections are highly prevalent in Belgian turkeys and therefore they both might contribute to the respiratory disease complex observed in turkeys. C. psittaci can infect turkeys within the first week of age, even in the presence of maternal antibodies. However, the first C. psittaci outbreaks occur mostly at the age of 3 to 6 weeks, the period when also E. coli infections appear on the farms. Therefore, we examined in this study the pathogenicity of an E. coli superinfection on C. psittaci predisposed turkeys. Turkeys were infected with C. psittaci, E. coli or with C. psittaci followed by E. coli. Simulating the impact of an E. coli infection during the acute phase or the latent phase of a C. psittaci infection, turkeys received E. coli at 1 or 5 weeks post C. psittaci infection, respectively. E. coli superinfection during the acute phase of C. psittaci infection increased C. psittaci excretion and stimulated chlamydial replication in the respiratory tract resulting in exacerbated clinical disease. Interestingly, E. coli superinfection during the latent phase of C. psittaci infection induced chlamydial replication, leading to increased C. psittaci-specific antibody titres. In addition, chlamydial predisposition gave higher E. coli excretion compared with turkeys that had only been infected with E. coli. Overall, the present study clearly demonstrates the pathogenic interplay between C. psittaci and E. coli resulting in more severe respiratory disease