RESUMO
Serratospiculiasis is a parasitic disease caused by filariid nematodes of the genus Serratospiculum that parasitise the air sacs of various species of falcons, bald eagles and Cooper's hawks around the world. An infection with Serratospiculum was recently confirmed in a nonspecific host, the great tit, in Slovakia. Parasitic material from this host was fixed for molecular analysis. Nematode found in the air sacs from a captive-bred gyrfalcon was also stored. Analysis of small subunit (18S) ribosomal DNA (18S rDNA) gene indicated that sequences from Serratospiculum sp. and Serratospiculoides amaculata were closely related to a reference sequence from Serratospiculum tendo, in agreement with morphology. This study is the first to generate molecular data and infer the phylogenetic position of S. amaculata as the first representative of the genus Serratospiculoides.
Assuntos
Sacos Aéreos/parasitologia , Doenças das Aves/parasitologia , Falconiformes/parasitologia , Infecções por Spirurida/veterinária , Espirurídios/classificação , Espirurídios/genética , Animais , Cruzamento , DNA Ribossômico , Feminino , Filogenia , RNA Ribossômico 18S/genética , Eslováquia , Espirurídios/isolamento & purificação , Infecções por Spirurida/parasitologiaRESUMO
BACKGROUND: Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). RESULTS: In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry. CONCLUSION: A proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.
Assuntos
Linfócitos B/imunologia , Camelídeos Americanos/imunologia , Imunização , Biblioteca de Peptídeos , Anticorpos de Domínio Único/biossíntese , Animais , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Bacteriófagos/genética , Técnicas de Visualização da Superfície Celular/economia , Técnicas de Visualização da Superfície Celular/métodos , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática , Imunização/economia , Imunização/métodos , Interleucina-2/imunologia , Interleucina-4/imunologia , Lipoproteínas/imunologia , Ativação Linfocitária , Anticorpos de Domínio Único/imunologiaRESUMO
West Nile virus (WNV) is a mosquito-borne neurotropic pathogen that presents a major public health concern. Information on WNV prevalence and circulation in Slovakia is insufficient. Oral and cloacal swabs and bird brain samples were tested for flavivirus RNA by RT-PCR using newly designed generic primers. The species designation was confirmed by sequencing. WNV was detected in swab and brain samples, whereas one brain sample was positive for tick-borne encephalitis virus (TBEV). The WNV sequences clustered with lineages 1 and 2. These results confirm the circulation of WNV in birds in Slovakia and emphasize the risk of infection of humans and horses.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Doenças das Aves/virologia , Aves/virologia , Primers do DNA/genética , Vetores de Doenças , Encefalite Transmitida por Carrapatos/transmissão , Cavalos , Humanos , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Eslováquia , Febre do Nilo Ocidental/transmissão , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/classificaçãoRESUMO
Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.