Evaluation of orf virus (ORFV) isolation in continuous lamb testis cells (OA3.Ts) and development of a co-culture method with infected cells to increase infectivity

Nashirudddullah, Nawab; Pathak, Devendra; Barman, Nagendra Nath; Ahmed, Jafrin Ara; Rajbongshi, Gitika; Kumar, Rajeev Ranjan

doi:10.18805/ijar.v0i0f.3786

Cited by 4 publications

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“…Besides the current applications, other confirmatory applications were also employed. In our earlier studies, ORFV has also been successfully cultured in continuous lamb testis cells (OA3.Ts) (Nashiruddullah et al, 2016) and the virus has been demonstrated by immunofluorescence and visualized by Transmission Electron Microscopy (TEM) (Nashiruddullah et al, 2018). ELISA applications for antibody detection showed positive results and were consistent.…”

Section: Dot-elisa For Antibody Detectionmentioning

confidence: 88%

“…Live attenuated vaccine virus (OrfMuk 59/05, batch 06/ 09) isolated from goat and previously propagated in primary lamb testis cells (PLT) produced at poxvirus disease laboratory, Division of Virology, Mukteshwar were used as a reference ORFV strain. Tissue lysates from scab samples were prepared according to Nashiruddullah et al (2016).…”

Section: Orfvirus (Orfv) Antigen Detectionmentioning

confidence: 99%

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Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Nashiruddullah¹,

Pathak²,

Barman³

et al. 2021

IJAR

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Background: The Study was intended to evaluate some common diagnostics that could supplement the clinical and histological identification of orf in goats.Methods: Samples from suspected clinical cases of orf (contagious ecthyma) were collected from various organized and unorganized goat herds around Guwahati, Assam. Presumptive diagnosis was based on the typical signs and lesions. For confirmatory diagnosis, various molecular and immunoassays were employed for the detection of orf virus or circulating antibodies.Result: Cutaneous lesions observed included solitary to multifocal erythema, papules, vesicles, pustules and scab stages on the lips, ears, gums, tongue, udder and perineal region. No mortality was observed and the morbidity rate varied between 35-60%. Microscopic lesions in skin biopsies were typically marked by epidermal hyperplasia and ballooning degeneration of the keratinocytes along with other changes. Eosinophilic intracytoplasmic inclusion bodies were demonstrable within keratinocytes only in early papulo-vesicular stages. Dermis was infiltrated with polymorphonuclear and mononuclear cells in varying proportions. Initial screening of samples was done with PCR systems to specifically detect parapoxvirus DNA employing a semi-nested PCR targeted against the partial B2L gene (with reported primers yielding 594 bp and 235 bp amplified products) and an uniplex PCR targeting the whole B2L gene (with reported primer set to yield an amplified product of 1206 bp). Agar gel immuno-diffusion (AGID) failed to develop positive immunoprecipitation with hyperimmune serum against scab lysates; however, counter-immuno-electrophoresis (CIE) showed positive immunoprecipitation in the same samples. For rapid and in situ detection of ORFV antigen in tissues, immunofluorescent and immunoperoxidase techniques were successfully employed both on cryosections and formalin fixed skin biopsies. Immunofluorescent technique on cryosections was found to be easy, rapid and more specific. A dot-ELISA was also developed for successful confirmation of orf virus from clinical samples. For antibody detection in convalescing goats, an indirect-ELISA and a dot-ELISA was also successfully tested to demonstrate for antibody detection in convalescing goats, but protective titres later after infection was not addressed. Monoclonal antibody employed in the assays was found to be specific, sensitive and versatile for virus detection from direct clinical samples. It is contemplated that assays employing hyperimmune sera or detection of circulating antibody against orf virus may have limited diagnostic applications owing to its partial and transient humoral immunity and the inherent property of the virus to modulate and interfere with the host response and evade immune mechanisms.

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Section: Dot-elisa For Antibody Detectionmentioning

confidence: 88%

Section: Orfvirus (Orfv) Antigen Detectionmentioning

confidence: 99%

Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Nashiruddullah¹,

Pathak²,

Barman³

et al. 2021

IJAR

Self Cite

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“…Formation of CPE due to virus growth was observed. TCID 50 values of the produced virus were determined by the Spermen-Kaerber method (Ergin and Köklü 1974;Nashirudddullah et al 2016).…”

Section: Detection Of Infective Titer Of Pathogen Ce Virusmentioning

confidence: 99%

“…At the end of the incubation period, 50 µl of MDBK cell culture was added to all wells and incubated in 5% CO 2 medium for 10 days at 37°C. Cells were checked daily for CPE formation (Nashirudddullah et al 2016).…”

Section: Serum Neutralization Test (Snt)mentioning

confidence: 99%

Contagious Ektima (CE) aşısının bağışıklık ve zararsızlık çalışmalarında tavşanların kullanılması

Gülyaz¹,

Saraç

Hasöksüz³

et al. 2020

Etlik Veteriner Mikrobiyoloji Dergisi

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Contagious ecthyma (CE) is a zoonotic viral infection and common localized cutaneous infection of young sheep and goats caused by a Parapoxvirus with worldwide distribution. The aim of this study was to determine whether rabbits will be used instead of lambs and kids in immune and safety studies after the production of CE vaccine. The titres of Pendik CE pathogen strain isolated from lamb (E(P)CK 4 ) and attenuated vaccine strain originated from lamb (E(P)CK 22 ) used in the study were TCID 50 10 6.50 and 10 7.00 /ml, respectively. In the study, to determine the pathogenicity of CE virus (E(P)CK 4 ) in rabbits, it was found that CE virus-specific hyperemia, vesicles and pustules were not observed and there was no increase in body temperature, and CE virus was not detected by PCR in the scabs located in the skin of back-waist regions. In the blood sera of rabbits treated with pathogen CE virus and vaccinated with CE vaccine virus, no antibodies were detected against CE virus. In the immunity study in lambs and kids vaccinated with CE vaccine (E(P)CK 22 ), it was found that the vesicles, pustules and scabs appeared on day 3 and the lesions healed on the 15 th day seen that CE vaccine virus protects lambs and kids against pathogen CE (E(P)CK 4 ) strain.

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“…CPE formation due to virus multiplication was observed. The titer of CE vaccine virus were determined according to the Spermen-Kaerber method (Ergin and Köklü 1974;Burleson et al 1992;Nashirudddullah et al 2016).…”

Section: Collection Of Ce Final Product (E (P) Ck 17 -Mdbk 5 )mentioning

confidence: 99%

Adaptation of Contagious Ecthyma vaccine strain to MDBK cell culture and immunity-stability studies in lambs

Gülyaz¹,

Saraç

Satir³

et al. 2020

Etlik Veteriner Mikrobiyoloji Dergisi

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Contagious ecthyma (CE) is a common viral infection in lambs and kids, and still maintains its importance in sheep and goat breeding. Attenuated, live and lyophilized CE vaccines adapted to cell culture are widely used in the fight against CE. The aim of this project was to adapt the CE vaccine strain to Madin Darby Bovine Kidney (MDBK) cell culture and to produce the vaccine, as well as determining the shelf life, stability, innocuity, and immune response in lambs.The titer of the vaccine virus adapted to the MDBK cell culture was determined to be TCID 50 10 6.5 / ml. In the innocuity study on mice and guinea pigs, no local or general reactions were observed. Body temperature of 10 lambs vaccinated by scarification was found to be between normal values (38.2-39.1°C). Neutralizing antibodies belonging to CE virus with VNT could not be detected in blood sera taken on 30 th day following the vaccination. As a result of challenge performed with pathogen (E(P)CK 5 ) CE virus, the protection was observed against CE infection in vaccinated lambs. In the stability study, the titers of lyophilized CE vaccine were found to be TCID 50 10 6.5 /ml from 1 to 15 months and 10 6.0 /ml at 18, 21 and 24 months during the 24-month storage period at +4/+8°C. It was determined that the vaccine virus remained as stable at TCID 50 10 6.5 /ml titer for 12 hours at 30, 33, 37 and 40°C and after 12 hours, the virus titer regressed to TCID 50 10 5.75 /ml at 42°C and 45°C. This study concluded that CE vaccines could be produced in MDBK cell cultures by adapting CE virus to MDBK cell culture.

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Evaluation of orf virus (ORFV) isolation in continuous lamb testis cells (OA3.Ts) and development of a co-culture method with infected cells to increase infectivity

Cited by 4 publications

References 0 publications

Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Contagious Ektima (CE) aşısının bağışıklık ve zararsızlık çalışmalarında tavşanların kullanılması

Adaptation of Contagious Ecthyma vaccine strain to MDBK cell culture and immunity-stability studies in lambs

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