Assessment of the pathogenicity of cell-culture-adapted Newcastle disease virus strain Komarov

Newcastle disease vaccines hitherto in vogue are produced from embryonated chicken eggs. Egg-adapted mesogenic vaccines possess several drawbacks such as paralysis and mortality in 2-week-old chicks and reduced egg production in the egg-laying flock. Owing to these possible drawbacks, we attempted to reduce the vaccine virulence for safe vaccination by adapting the virus in a chicken embryo fibroblast cell culture (CEFCC) system. Eighteen passages were carried out by CEFCC, and the pathogenicity was assessed on the basis of the mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index, at equal passage intervals. Although the reduction in virulence demonstrated with increasing passage levels in CEFCC was encouraging, 20% of the 2-week-old birds showed paralytic symptoms with the virus vaccine from the 18th(final) passage. Thus, a tissue-culture-adapted vaccine would demand a few more passages by CEFCC in order to achieve a complete reduction in virulence for use as a safe and effective vaccine, especially among younger chicks. Moreover, it can be safely administered even to unprimed 8-week-old birds.(AU)

Oral immunization of mice with attenuated Salmonella typhimurium vaccine expressing transferrin-binding protein A (TbpA) of Haemophilus parasuis

Background: Haemophilus parasuis is the etiological agent responsible for causing Glässers disease in pigs, which aremajor respiratory pathogens that cause large economic losses in the pig industry worldwide. H. parasuis obtains transferrinbound iron by expressing two surface receptors, transferrin-binding protein A and B (TbpA and B). The TbpA and B arecapable of binding to transferrin, and an impairment of iron uptake mechanisms is likely to induce virulence. For thisreason, these proteins can be useful as a candidate target for H. parasuis vaccination. Also, the live attenuated SalmonellaTyphimurium expressing recombinant antigens from other pathogens are attractive vaccine vectors.Materials, Methods & Results: In this study, we constructed attenuated S. Typhimurium vaccine strain by porcine neurtophilpassage method. By the passage, the ability of the neutrophil-adapted isolate to utilize d-xylose was lost, while the abilityof the strain to ferment trehalose was delayed after 2 or more days of the culture. The aspartate β-semialdehyde dehydrogenase (asd) gene was eliminated from S. Typhimurium by one-step PCR. Deletion of asd region was confirmed by PCRusing primers specific to the endpoints of the targeted region. TbpA fragment was amplified by PCR from genomic DNAof H. parasuis serotype 5. To construct TbpA expression plasmids, tbpA was subcloned downstream from the β-lactamasesignal sequence in the multicopy asd+ pYA3493 vector. This plasmid was subsequently electrotransformed into attenuatedS. Typhimurium. The 636bp fragment of the tbpA gene of H. parasuis in attenuated S. Typhimurium was amplified by PCRand the in-frame fusion of the tbpA was confirmed by nucleotide sequencing. The used this strain with Asd+ balanced-lethalplasmid pYA3493 vector to specify recombinant TbpA antigen, conserved immunogenic region of H. parasuis. Expression of the TbpA protein was analyzed by sodium dodecyl sulfate-polyacrylamide...(AU)

Oral immunization of mice with attenuated Salmonella typhimurium vaccine expressing transferrin-binding protein A (TbpA) of Haemophilus parasuis

Background: Haemophilus parasuis is the etiological agent responsible for causing Glässer’s disease in pigs, which aremajor respiratory pathogens that cause large economic losses in the pig industry worldwide. H. parasuis obtains transferrinbound iron by expressing two surface receptors, transferrin-binding protein A and B (TbpA and B). The TbpA and B arecapable of binding to transferrin, and an impairment of iron uptake mechanisms is likely to induce virulence. For thisreason, these proteins can be useful as a candidate target for H. parasuis vaccination. Also, the live attenuated SalmonellaTyphimurium expressing recombinant antigens from other pathogens are attractive vaccine vectors.Materials, Methods & Results: In this study, we constructed attenuated S. Typhimurium vaccine strain by porcine neurtophilpassage method. By the passage, the ability of the neutrophil-adapted isolate to utilize d-xylose was lost, while the abilityof the strain to ferment trehalose was delayed after 2 or more days of the culture. The aspartate β-semialdehyde dehydrogenase (asd) gene was eliminated from S. Typhimurium by one-step PCR. Deletion of asd region was confirmed by PCRusing primers specific to the endpoints of the targeted region. TbpA fragment was amplified by PCR from genomic DNAof H. parasuis serotype 5. To construct TbpA expression plasmids, tbpA was subcloned downstream from the β-lactamasesignal sequence in the multicopy asd+ pYA3493 vector. This plasmid was subsequently electrotransformed into attenuatedS. Typhimurium. The 636bp fragment of the tbpA gene of H. parasuis in attenuated S. Typhimurium was amplified by PCRand the in-frame fusion of the tbpA was confirmed by nucleotide sequencing. The used this strain with Asd+ balanced-lethalplasmid pYA3493 vector to specify recombinant TbpA antigen, conserved immunogenic region of H. parasuis. Expression of the TbpA protein was analyzed by sodium dodecyl sulfate-polyacrylamide...

Live bacterial vaccine vectors: An overview

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.

A hematologic and electrophoretic study in puppies vaccinated against canine distemper virus and canine parvovirus

Background: Canine distemper is a contagious multisystemic viral disease that affects canines and others carnivores. Canine parvovirus infection is one of the most important viral diseases in young dogs. Side effects of vaccine generally include fever, lethargy and local inflammation. Complementary exams are important to evaluate the strenght of immungenic stimulation. This study was aimed at evaluating hematological and electrophoretic alterations in puppies after inoculation of live attenuated vaccine against canine distemper virus and canine parvovirus. Materials, Methods & Results: Five non-breeding newborn dogs of the same litter were used. Animals received three subcutaneous injection of 1mL (at days 0, 21 and 42). Blood was collected at day 0 (day of vaccination) and for three times for each dose: at days 7, 14 and 21 (first dose); at days 28, 35 and 42 (second dose); and at days 49, 56 and 63 (third dose). Blood containing ethylenediaminetetraacetic acid as anticoagulant was used for hematological evaluation. The total serum protein were determined by the biuret method, using commercial reagent, according to fabricant instructions. Serum was used for protein fractionation by using cellulose acetate strip electrophoresis. A decrease in platelet count was observed at days 7 and 28 post-vaccination. Lymphocyte number increased 88.4%, as well as the level of the protein fractions alpha-1 globulin (68%) and alpha-2 globulin (41.4%) at day 7. Moreover, a 5-fold increase in the fibrinogem concentration and in the number of eosinophils was observed at day 14. Thereafter, the platelet count decreased by 27.3% and the number of monocytes increased 5-fold at day 28. Discussion: Mild to moderate thrombocytopenia is often observed in dogs 3-5 days post-vaccination with live attenuated vaccines, mainly those against CDV and CPV. Besides the platelet damage caused by the CDV per se, infected animals showed secondary immune-mediated thrombocytopenia and decreased platelet production due to direct viral megakaryocyte infection. The increase in alpha-1 globulin may be related to the augment in the synthesis of alpha-1 antitrypsin, the main protein of the alpha-1 globulin region, in response to the vaccine-induced acute inflammatory process. The alpha-2 globulin region includes haptoglobin, alpha-2 macroglobulin and ceruloplasmin, and the increase observed in this fraction suggested that both haptoglobin and ceruloplasmin levels were augmented, following acute inflammatory response pattern. Fibrinogen is a soluble plasma glycoprotein that is converted by thrombin into fibrin during blood clotting. Despite the increase in fibrinogen concentration be the best indicator of inflammation in large animals, the hyperfibrinogenemia observed suggests that the inflammatory process was adequate to stimulate synthesis of this acute phase protein (P < 0,05). Absence of lymphocytosis observed at days 49, 56 and 63 associated to the progressive increase of the gamma globulin fraction, although not statistically significant, suggested an augment of B lymphocytes. The eosinophilia was observed in highlighting the presence of inflammation. Moreover, an increase in monocyte count indicating the presence of subacute or chronic inflammation after the second dose of the vaccine.

Caracterização de amostras atenuadas do vírus da Diarréia Viral Bovina (BVDV) tipos 1 e 2 para uso em vacinas / Characterization of bovine viral diarrhea virus (BVDV) types 1 and 2 isolates for use in vaccines

Este artigo relata a caracterização de duas amostras citopáticas do vírus da Diarréia Viral Bovina (BVDV-1: IBSP-2; BVDV-2: SV-253) submetidas à atenuação por múltiplas passagens em cultivo celular e exposição à radiação ultravioleta. As amostras foram caracterizadas in vitro (tamanho e morfologia de placas, cinética de replicação e perfil antigênico) e in vivo (atenuação e resposta sorológica em bovinos). A caracterização in vitro de populações clonadas dos vírus obtidas nas diferentes passagens em cultivo celular (0, 1, 10, 20 e 30), demonstrou que o processo de atenuação não afetou negativamente as características antigênicas e fenotípicas das amostras. Não foram observadas alterações significativas no tamanho e morfologia de placas e na cinética de replicação. A reatividade com 48 anticorpos monoclonais demonstrou que o perfil antigênico não doi alterado durante as sucessivas passagens in vitro. A inoculação intramuscular dos clones de vírus obtidos na passagem 30 (IBSP-2: 10(7,3) DICC50; SV-253: 10(6,8) DICC50) em 12 novilhas soronegativas com idade média de 15 meses, não resultou em sinais clínicos, comprovando sua atenuação. Após a inoculação, o vírus foi detectado em leucócitos da maioria dos animais inoculados (10/12) entre os dias 3 e 6 pós-inoculação (pi) e em secreções nasais de três animais (dias 4, 7 e 8pi). No entanto, não ocorreu transmissão dos vírus vacinais aos três animais soronegativos mantidos como sentinelas. Todos os animais vacinados soroconverteram aos 14 dias pós-vacinação (dpv). Títulos moderados a altos de anticorpos neutralizantes foram detectados frente a 5 isolados brasileiros do BVDV-1 (títulos de 80 a > 1280) e quatro isolados do BVDV-2 (títulos de 20 a 640). Em geral, os títulos foram de magnitude superior frente a isolados brasileiros do BVDV-1. Aos 240dpv, os animais receberam uma segunda dose dos vírus vacinais (IBSP-2: 10(7,3) DICC50; SV-253: 10(6,8) DICC50) ...(AU)

This article reports the characterization of two cytopathic isolates of bovine viral diarrhea virus (BVDV-1: IBSP-2; BVDV-2:SV-253) submitted to multiple passages (n=30) in tissue culture associated with ultraviolet irradiation. The vaccine candidate strains were characterized in vitro (plaque size and morphology, growth kinetics and antigenic profile) and in vivo (attenuation and serological response in calves). In vitro characterization of biologically cloned viruses obtained at passages 0, 1, 10, 20 and 30 demonstrated that the attenuation process did not significantly affect the phenotypic and antigenic properties of the viruses. No major differences in plaque size and morphology and in the growth kinetics in tissue culture were observed among the viruses obtained at different passages. Likewise, the antigenic profile of these viruses did not change upon successive passages in tissue culture, as ascertained by the pattern of binding by 48 monoclonal antibodies (mAbs). Intramuscular inoculation of both viruses (IBSP-2: 107.3 TCID50; SV-253: 106.8 TCID50) at passage 30 (p30) in twelve 15 months old heifers did not produce clinical signs, demonstrating the attenuation of the viruses. Following inoculation, infectious virus was detected in leucocytes of most inoculated animals (10/12) between days 3 and 6 post-inoculation (pi) and in nasal secretions of three animals (days 4, 7 and 8pi). However, the vaccine viruses were not transmitted to three seronegative calves maintained as sentinels. All vaccinated calves seroconverted at day 14 post-vaccination. A moderate to high serum neutralizing response against five Brazilian BVDV-1 (titers from 80 to > 1,280) and four Brazilian BVDV-2 isolates (titers from 20 to 640) was observed at day 33 post-vaccination (pv). In general, the highest titers were observed against the Brazilian BVDV-1 isolates ... (AU)