Taqman-based Reverse Transcription and Real-time PCR for Diagnosis of Peste des Petits Ruminants (PPR) /Small Ruminant Morbilli (SRM) Virus from Clinically Affected Animal Samples Collected in Thrissur District, Kerala State and Molecular Epidemiology of Recovered Isolates |Chapter 9 | Innovations in Biological Science Vol. 4

 

Peste des petits ruminants (PPR) is caused by a virus of the family Paramyxoviridae, genus Morbillivirus and species Morbillivirus caprinae or small ruminant Morbilli virus. PPR also known as ovine rinderpest is an acute, highly contagious, World Organization for Animal Health (WOAH) notifiable and economically important transboundary viral disease affecting primarily sheep and goats associated with high morbidity and mortality. Camels and wild small ruminants can also be affected by PPRV.  PPR is considered as one of the main constraints in augmenting the productivity of small ruminants in developing countries and severely affects poor farmer’s economies. The disease is clinically manifested by pyrexia, occulo-nasal discharges, necrotizing and erosive stomatitis, gastroenteritis, diarrhea, bronchopneumonia and can be diagnosed from classic clinical signs, pathological lesions, and specific detection of virus antigen-surface proteins/antibodies/viral RNA in the clinical samples by various immune chromatographic/serological tests and molecular assays. The current diagnostic strategy for PPR used in network laboratories of AHD Kerala is mainly based on serological assays.  The present study aimed to standardize Taqman-based reverse transcription Real-Time PCR (RT PCR) protocols for the detection of PPR virus in samples like EDTA blood collected during viraemic/febrile phase and tissue samples after post-mortem from clinically affected animals- and to study the molecular epidemiology of isolates recovered targeting Fusion protein (F) gene.

 

 

Author(s) Details

Sunitha Karunakaran

Animal Disease Control Project, Thrissur, India and Veterinary Dispensary, Koorkanchery, Thrissur, India.

Beena. P. S

Omicsgen Life Sciences Limited, North Kalamassery, Kochi, Kerala, India.

Gopika Thattat Gopinathan

Department of Animal Husbandry, Clinical Laboratory, Animal Disease Control Project, Thrissur, Kerala, India.

Hareesh Sasidharan

Animal Disease Control Project, Thrissur, India and Veterinary Dispensary, Koorkanchery, Thrissur, India.

 

Please see the book here:- https://doi.org/10.9734/bpi/ibs/v4/11456F

TaqMan-Based Real-Time PCR for Diagnosis of Multiple Tick-Transmitted Diseases among the Bovine Population in Thrissur and Wayanad Districts, Kerala State, India | Chapter 3 | Microbiology and Biotechnology Research: An Overview Vol. 4

TaqMan PCR (Polymerase Chain Reaction) is a type of real-time PCR, and it uses a nucleic-acid probe complementary to an internal segment of the target DNA. The present study aimed to use TaqMan-based qualitative Real Time PCR (qPCR) as an aid in accurate and confirmatory diagnosis up to species level for multiple tick-transmitted pathogens affecting the bovine population. Conventional diagnostic methods based on microscopy will not be sufficient to identify multiple tick-transmitted pathogens, pathogens having similar infective stages and their identification up to species level. A total of 60 peripheral blood samples in EDTA were collected randomly from five different private cattle farms in Wayanad and Thrissur districts, Kerala state. Peripheral blood in EDTA was stored at-80°C until usage and processed for preparation of template DNA as per the protocols described in the High Pure PCR template preparation kit. Primer efficiency testing was conducted by plotting a standard curve. Custom-synthesised positive clone in pUGM plasmid (with initial concentration 95ng/µl for A. phagocytophilum, and 10ng/µl for others) was used for primer efficiency testing and standardisation of cycling conditions. The average Ct value observed for 38 positive samples for the Theileria genus was 12.79 cycles, ranging from 4.1 to 36.19 cycles. The average Ct value observed for 31 positive samples for M. haemocanis was 22.42 cycles, ranging from 2.66 to 37.6 cycles. The average Ct value observed for 22 positive samples for P. multocida was 20.20 cycles, ranging from 2.74 to 35.72 cycles. Lower Ct/Cq values corresponded to a greater amount of initial template. In most of the tick-transmitted diseases among the bovine population presented for treatment, P. multocida is causing severe problems as a secondary pathogen; the reason may be attributed to suppression of adaptive immune response by the primary tick-transmitted pathogens. This data should give an insight while planning mass vaccination strategies for bovine population in the state of Kerala; by Department of Animal Husbandry because a cock tail of tick transmitted pathogens in a single animal will interfere with the future sero conversion and duration of immunity.

 

Author(s) Details

 Sunitha Karunakaran
Department of Animal Husbandry, Clinical Laboratory, Animal Disease Control Project Paravattani, Thrissur, East P.O-680005, Kerala, India.

Hareesh Sasidharan
Department of Animal Husbandry, Clinical Laboratory, Animal Disease Control Project Paravattani, Thrissur, East P.O-680005, Kerala, India.

 

Gopika Thattat Gopinathan
Department of Animal Husbandry, Clinical Laboratory, Animal Disease Control Project Paravattani, Thrissur, East P.O-680005, Kerala, India.

 

Please see the book here:- https://doi.org/10.9734/bpi/mbrao/v4/5690

Taqman-based Reverse Transcription and Real-time PCR for Diagnosis of Peste des Petits Ruminants (PPR) /Small Ruminant Morbilli (SRM) Virus from Clinically Affected Animal Samples Collected in Thrissur District, Kerala State and Molecular Epidemiology of Recovered Isolates | Chapter 9 | Innovations in Biological Science Vol. 4

Peste des petits ruminants (PPR) is caused by a virus of the family Paramyxoviridae, genus Morbillivirus and species Morbillivirus caprinae or small ruminant Morbilli virus. PPR also known as ovine rinderpest is an acute, highly contagious, World Organization for Animal Health (WOAH) notifiable and economically important transboundary viral disease affecting primarily sheep and goats associated with high morbidity and mortality. Camels and wild small ruminants can also be affected by PPRV.  PPR is considered as one of the main constraints in augmenting the productivity of small ruminants in developing countries and severely affects poor farmer’s economies. The disease is clinically manifested by pyrexia, occulo-nasal discharges, necrotizing and erosive stomatitis, gastroenteritis, diarrhea, bronchopneumonia and can be diagnosed from classic clinical signs, pathological lesions, and specific detection of virus antigen-surface proteins/antibodies/viral RNA in the clinical samples by various immune chromatographic/serological tests and molecular assays. The current diagnostic strategy for PPR used in network laboratories of AHD Kerala is mainly based on serological assays.  The present study aimed to standardize Taqman-based reverse transcription Real-Time PCR (RT PCR) protocols for the detection of PPR virus in samples like EDTA blood collected during viraemic/febrile phase and tissue samples after post-mortem from clinically affected animals- and to study the molecular epidemiology of isolates recovered targeting Fusion protein (F) gene.

Author(s) Details:

Sunitha Karunakaran,
Animal Disease Control Project, Thrissur, India and Veterinary Dispensary, Koorkanchery, Thrissur, India.

Beena. P. S
Omicsgen Life Sciences Limited, North Kalamassery, Kochi, Kerala, India.

Gopika Thattat Gopinathan
Department of Animal Husbandry, Clinical Laboratory, Animal Disease Control Project, Thrissur, Kerala, India.

Hareesh Sasidharan
Animal Disease Control Project, Thrissur, India and Veterinary Dispensary, Koorkanchery, Thrissur, India.

 

Please see the link here: https://stm.bookpi.org/IBS-V4/article/view/14398