Comparative efficacy of different Carbopol concentrations as a stabilizer of live attenuated sheep pox virus vaccine against lumpy skin disease

In Egypt, the lyophilized live attenuated sheep pox virus vaccine has been used for the vaccination of cattle against lumpy skin disease virus to control its economic impact on livestock industry. In this endeavor, we validate the efficacy of Carbopol® as a stabilizer and adjuvant to enhance immunogenicity of such a heterologous sheep pox virus vaccine against lumpy skin disease. Lyophilization of sheep pox virus vaccine stabilized with Carbopol® produced better physical and antigenic properties than freeze-drying with lactalbumin/sucrose stabilizer; this was manifested by superior disc uniformity, thermo-stability at 37oC, and less reduction in virus titer. Immunization of calves' groups with variable sheep pox vaccine doses containing different Carbopol® concentrations revealed that 103.5 TCID50 of sheep pox virus vaccine enclosing 0.5 percent Carbopol® is the field dose of choice. Moreover, it induced protective serum neutralizing index of 2.5 and a ELISA S/P ratio of 36, by the 4th week post vaccination. Besides, the inclusion of 0.5 percent Carbopol® in formulation of the sheep pox virus vaccine was safe in bovines and enhanced cellular immune response to lumpy skin disease virus, as evidenced by increased T cell proliferation. Hence, it is recommended to use Carbopol® as 0.5 percent in preparation of live attenuated sheep pox virus vaccine to confer better protection against lumpy skin disease virus infection(AU)

En Egipto, la vacuna atenuada liofilizada contra el virus de la viruela ovina ha sido utilizado para la vacunación del ganado, contra el virus de la dermatosis nodular contagiosa, para controlar su impacto económico en la industria ganadera. En este trabajo, validamos la eficacia del Carbopol®, como estabilizador y adyuvante, para mejorar la inmunogenicidad de dicha vacuna heteróloga contra la dermatosis nodular contagiosa. La liofilización de la vacuna contra el virus de la viruela ovina estabilizada con Carbopol®, resultó en mejores propiedades físicas y antigénicas que la liofilización con el estabilizador de lactoalbúmina/sacarosa; lo anterior se manifestó en la uniformidad superior del disco, la termoestabilidad a 37°C y la menor reducción del título del virus. La inmunización de grupos de terneros con dosis variables de vacuna contra el virus de la viruela ovina, que contenían diferentes concentraciones de Carbopol®, reveló que la dosis de campo de elección fue 103,5 TCID50 de la vacuna contra el virus de la viruela ovina conteniendo 0,5 por ciento de Carbopol®, la que indujo un índice de neutralización sérica protectora de 2,5 y una relación S/P de ELISA de 36 a la cuarta semana después de la vacunación. Además, la inclusión de Carbopol® al 0,5 por ciento en la formulación de la vacuna contra el virus de la viruela ovina fue segura en los bovinos y potenció la respuesta inmunitaria celular contra el virus de la dermatosis nodular contagiosa, como lo demuestra el aumento de la proliferación de células T. Por lo tanto, se recomienda el uso de Carbopol® al 0,5 por ciento en la preparación de la vacuna viva atenuada contra el virus de la viruela ovina para conferir una mejor protección contra la infección por el virus de la dermatosis nodular contagiosa(AU)

Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus / 生物工程学报

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β2m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a SuperdexTM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.

new approach to develop a vaccine against capripox infection in sheep and goats using a new strain of sheep pox virus in Iran

This report describes the preparation of a safe and immunogenic single vaccine against capripox disease in sheep and goats using two different types of cells [primary lamb kidney and vero, a bovine turbinate cell line]. This used the 0240 pox strain isolated from sheep during an outbreak. The prepared vaccine was administered subcutaneously and induced complete protection against an experimental sheep and goat pox challenge virus. For the final evaluation, the efficacy of the prepared vaccine was used in a field trial under the supervision of veterinary clinicians. This vaccine was proved to be more efficacious than the routine vaccines that are currently produced

Immunomodulating effect of ampicillin and cephalosporin in sheep vaccinated with sheep poxvirus vaccine

In this study we use 20 sheep divided into four groups [one group was injected with the vaccine, the second was taken the vaccine and ampicillin, the third group was taken the vaccine and cephalosporin and the fourth act as a control]. The immunity was measured humoral immunity to sheep pox virus vaccine by neutralization test and cell mediated immunity by lymphocyte transformation assay as well as phagocytosis. The results revealed that an increase of the immune status in group injected with antibiotics with vaccine than that received the vaccine only and than the control group. There were an increase of lymphocyte transformation test, neutralization test and phagocytosis. It could be concluded that ampicillin and cephalosporin directly increase the immune status in sheep vaccinated with sheep poxvirus vaccine

Recombinant goat pox virus expressing PPRV H protein / 生物工程学报

The purpose of the study is to construct recombinant goat pox virus (GPV) expressing Peste des petits ruminants virus (PPRV) H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene (rGPV-PPRV-H) was selected and purified by gpt and eGFP utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express H protein of PPRV while infecting lamb testis cells. Six goats were immunized with 2 x 10(6) PFU rGPV-PPRV-H through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80, > or = 80, > or = 80, 40, > or = 80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all > or = 80, and the neutralization antibodies of PPRV were > 80, 80, > 80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine.

Development and application of TaqMan-MGB real-time quantitative PCR assay for detection of goat pox virus / 生物工程学报

The complete gene sequences of eight capripoxvirus strains in GenBank were aligned and analyzed with DNAStar software. We selected a size of 64 bp gene fragment that was located in gp064 region of goat pox virus (GPV) genome, and designed a pair of primers and a TaqMan-MGB probe against the gene fragment with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed and the standard curve of different dilution series was described. We extracted the DNA samples from clinical skin pox, scab and GPV infected materials of artificial challenge animals. The FQ-PCR assay has been performed for all kinds of DNA samples. The results showed that the FQ-PCR assay was sensitive, specific, stable and could be used for clinical diagnosis. This method provided an important tool for rapid diagnosis of goat pox clinically, and for study GPV pathogenesis in the course of disease occurrence, development and convalescence.

Cell culture adapted sheeppox virus as a challenge virus for potency testing of sheeppox vaccine.

Sheeppox virus from an outbreak of sheeppox that occurred in Srinagar (Jammu and Kashmir, India) in 2000 was isolated by inoculation of susceptible sheep and further re-isolated in cell culture. The field virus, adapted to grow in lamb testes culture, was evaluated for its potential use as challenge virus in potency testing of sheeppox vaccine currently in use. The virus (passage 6) produced severe disease in susceptible sheep when inoculated subcutaneously with a dose of 106.2 TCID50. The virus identity was confirmed by PCR, sequencing of P32 gene and species-specific signature residues identified in deduced aa sequence of the gene. The virus was successfully evaluated for its virulence using two batches of sheep pox vaccines. Use of this field virus enables consistent potency experiments of sheeppox vaccines avoiding use of animals for its propagation and titration.

In vitro antiviral activity of plant extracts on goatpox virus replication.

Four plants having known medicinal properties were screened for inhibition of goatpox virus (GTPV) replication in vitro. Of the 4 plants, extract of Acacia arabica (Babul) and Eugenia jambolana (Jamun) leaves had inhibition (%) 99.70 and 99.92 at their maximum non toxic concentrations, 99.93 +/- 0.38 and 1999.73 +/- 0.50 microg/ml, respectively in all cytopathic effect (CPE) inhibition assays. Inhibition of GTPV virus replication was further confirmed by PCR and SYBR Green based quantitative real-time QPCR assays specific for GTPV. Results indicated that the extract of Acacia arabica and Eugenia jambolana leaves inhibited GTPV replication in vitro.

Construction, expression and immunogenicity of eukaryotic vectors based on goat pox virus P32 gene / 病毒学报

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.

Comparação in vitro de duas técnicas de fixação interna rígida utilizadas em avanços mandibulares: estudo biomecânico em mandíbulas de carneiro / In vitro comparison of two techniques of rigid internal fixation used for mandibular advancements: biomechanical study in sheep mandible

Os principais métodos para a fixação da osteotomia sagital do ramo mandibular são a utilização de placas monocorticais de forma não compressiva e a instalação de parafusos bicorticais de maneira posicional, cada um deles apresentando vantagens e desvantagens. Ambos os métodos são descritos como rígido e, dessa forma, eliminam a necessidadde de bloqueio maxilomandibular no pós-operatório, contudo, existem dois pontos a serem esclarecidos: o primeiro é se ambas as formas de fixação promovem a mesma rigidez e o segundo é o quanto de carga cada forma de fixação pode tolerar. O resultado obtido foi que a força necessária para levar o sistema a falhar é muito maior no caso da fixação realizada por meio de parafusos bicorticais, ou seja, este tipo de fixação apresenta uma resistência mecânica maior

Avaliaçäo de dois sistemas para coleta de sangue de carneiro utilizado no preparo de meios de cultura / Evaluation of two systems for sheep blood collection used for preparing culture media

Hemoderivados de origem animal säo amplamente empregados no preparo de meios de cultura utilizados em práticas laboratoriais de interesse em Saúde Pública. Para avaliaçäo da qualidade deste produto foram testados dois sistemas de coleta de sangue em carneiro. O sistema tradicional, utilizando seringa, e um sistema em bolsa confeccionada em cloreto de polivinila (PVC) atóxico. Foram utilizados 40 carneiros adultos, machos e fêmeas, distribuídos de forma aleatória em dois grupos compostos por vinte animais cada. O sangue coletado para o estudo foi manipulado com assepsia em ambiente estéril, sendo posteriormente incubado a 37 graus C durante 24 horas e semeado em placas com ágar Mueller Hinton sangue (MHS) e em tubos com caldo de infusäo cérebro coraçäo (BHI). Os dados obtidos demonstraram a conveniência do uso da bolsa em PVC, por näo apresentar crescimento bacteriano em nenhuma das amostras testadas

Animal-origin hemoderived products are largely employed for culture media that accountfor laboratory practice of interest for Public Health. In order to evaluate the quality of this product, twosystems of blood collection were tested, the traditional system that makes use of syringe, and a bagsystem made of atoxic polyvinyl chloride (PVC). Forty adult sheep, males and females, distributed atrandom in two groups of twenty animals each, were used. Blood used in this study was collected withasepsis, in a sterile environment, incubated at 37°C for 24 hours, and then streaked out on Mueller Hintonblood agar plates and seeded in tubes with Brain Heart Infusion Broth (BHI). Our data showed that thePVC bags did not present any bacterial growth in the tested samples, indicating to be better than thetraditional system

Immunopharmacological and toxicological effect of ofloxacin in sheep

In this study, the effect of ofloxacin on the immune response was investigated in sheep vaccinated with the live attenuated sheep pox virus vaccine. The evaluation of the cellular immune response revealed a significant decrease in total leucocytic count, lymphocyte blastogenesis, phagocytosis% and bacterial killing% in groups administered 10 and 20 mg ofloxacin/kg b. wt. once daily for five successive days either simultaneously with sheep pox virus vaccine or one week post vaccination. The administration of ofloxacin in therapeutic [10 mg/kg b. wt.] and double therapeutic [20 mg/kg b. wt.] doses either simultaneously with sheep pox virus vaccine or one week post vaccination produced a significant reduction in antibody titers, total serum proteins, globulins and albumin. The biochemical assay of sera revealed a significant decrease in the level of packed cell volume, hemoglobin percentage and iron in ofloxacin treated groups either simultaneously with sheep pox virus vaccine or one week post vaccination. However, ofloxacin treatment five days prior to vaccination evoked a insignificant variation in cellular and humoral immune response