Trop Anim Health Prod (2015) 47:247–249 DOI 10.1007/s11250-014-0707-1
SHORT COMMUNICATIONS
Detection of peste des petits ruminants virus in formalin-fixed tissues Simon Mwangi Kihu & George Chege Gitao & Lily Caroline Bebora & Munene John Njenga & Gidraph Gachunga Wairire & Ndichu Maingi & Raphael Githaiga Wahome & Julius Otieno Oyugi & Ernest Lutomia
Received: 31 July 2014 / Accepted: 8 October 2014 / Published online: 19 October 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Peste des petits ruminants virus that causes a highly infectious and often fatal disease of sheep and goats is confirmed by various diagnostic techniques among them being isolation of the virus from cell culture systems, viral ribonucleic acid (RNA) detection by molecular assays, and viral antigen detection by immunocapture enzyme-linked immunosorbent assay (IC ELISA), immunohistochemistry (IHC), and AGAR gel test. Whereas most of the confirmatory diagnostic procedures require pathological samples to be stored frozen to preserve integrity of the peste des petits ruminants (PPR) virus RNA, samples for IHC tests are preserved in 10 % formalin. In this study, nine formalin-fixed pathological samples from three goats suspected of PPR were processed for extraction of PPR viral RNA and analyzed for detection with real-time reverse transcription–polymerase chain reaction (qRT-PCR) assay. The results showed that five out of the nine tested samples returned positive for presences PPR viral genome. This study has established that field pathological samples of PPR-suspected cases, collected and stored in 10 % formalin for up 2 years, could be used for PPR virus RNA extraction for disease virus confirmation. Keywords Peste des petits ruminants virus . Ribonucleic acid . Formalin-fixed tissues S. M. Kihu (*) : G. C. Gitao : L. C. Bebora : M. J. Njenga : N. Maingi : R. G. Wahome Faculty of Veterinary Medicine, University of Nairobi, P.O. Box 29053, 00625 Uthiru, Kenya e-mail: [email protected] G. G. Wairire Faculty of Arts, University of Nairobi, P.O. Box 30197, 00100 Nairobi, Kenya J. O. Oyugi : E. Lutomia University of Nairobi Institute of Tropical and Infectious Diseases (UNITID), P.O. Box 19676, 00202 Nairobi, Kenya
Introduction Peste des petits ruminants (PPR) is a highly infectious and often fatal disease of sheep, goats, and wild small ruminants that is caused by peste des petits ruminants virus (PPRV) classified under genus Morbillivirus in the family Paramyxoviridae (Libeau et al. 2014). PPR disease outbreaks can cause mortality as high as 90 % in immunologically naive sheep and goat populations resulting in significant negative impact to food security and pastoral economies of subsistence farmers who are dependent on small ruminants particularly in the developing countries of the tropical region (Libeau et al. 2014). Peste des petits ruminants is currently confined to Middle East, South and central Asia, China, and Africa regions that host most of the developing countries (Munir et al. 2013). Effective diagnosis and eventual control of PPR is thus considered an essential element in the fight for global food security and poverty alleviation in these developing countries (Dundon et al. 2014). Tentative diagnosis of PPR is done through observation of clinical disease and serology, whereas confirmatory diagnosis of PPR is done through various diagnostic techniques among them laboratory isolation of PPR virus in cell culture, detection of viral antigen by immunocapture enzyme-linked immunosorbent assay (IC ELISA), immunohistochemistry (IHC), and AGAR gel test, and molecular detection of PPR viral RNA using real-time reverse transcription–polymerase chain reaction (qRT-PCR) assay (Troung et al. 2014; Munir et al. 2012; Aytekin et al. 2011; Adombi et al. 2011). Apart from IHC, the other confirmatory diagnostic procedures require pathological samples to be stored frozen to preserve the integrity of the PPR virus RNA thus yielding high-quality RNA during the extraction process. In situations where samples are preserved as formalin-fixed tissues and are to be used in extraction of viral RNA, the quality and quantity of RNA extracted is dependent on the initial process of fixation and
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the extent of reversal of crosslinks formed by proteins and nucleic acids involving hydroxymethylene bridges of formalin that confound extraction of intact RNA from formalinfixed tissues (Scicchitano et al. 2006). A comparative study of methods for RNA extraction from formalin-fixed tissues reported by Sharma et al. (2012) concluded that formalinfixed tissues can be used for RNA extraction to be used for gene expression, pathogen detection, and epidemiological studies. In this study, an attempt is made to extract and detect PPR viral RNA from formalin-fixed tissues collected from field cases of PPR disease.
Materials and methods The study samples were collected from Lotakaa village herd in Turkana, Kenya (N 03° 38 390; S 034° 50 987) in 2011. Nine laboratory samples that included lung tissues and mesenteric and mediastinal lymph nodes; and colons were collected from carcasses and preserved in 10 % formalin. Extraction of PPR RNA was as outlined by RNeasy® FFPE kit protocol supplied by QIAGEN®. The extracted RNA samples were analyzed in a MicroAmp® Optical 96-well reaction plate by Abiprism® 7500 (Applied Biosystems) thermocycler using a specific qRT-PCR assay called TaqVet™ Peste des Petits Ruminants Virus kit which had a set of primers/probes designed from the Nterminus of the N-gene in the Mix PPRV based on a protocol supplied by manufacturer. The forward primer matched positions 483>508 in the (5-AGAGTTCAATATGTTRTTAGCC TCCAT-3); the TaqMan® probe positions 551>576 (FAM-5CACCGGAYACKGCAGCTGACTCAGAA-3- MGB) and the reverse primer were located at positions 603
SHORT COMMUNICATIONS
Detection of peste des petits ruminants virus in formalin-fixed tissues Simon Mwangi Kihu & George Chege Gitao & Lily Caroline Bebora & Munene John Njenga & Gidraph Gachunga Wairire & Ndichu Maingi & Raphael Githaiga Wahome & Julius Otieno Oyugi & Ernest Lutomia
Received: 31 July 2014 / Accepted: 8 October 2014 / Published online: 19 October 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Peste des petits ruminants virus that causes a highly infectious and often fatal disease of sheep and goats is confirmed by various diagnostic techniques among them being isolation of the virus from cell culture systems, viral ribonucleic acid (RNA) detection by molecular assays, and viral antigen detection by immunocapture enzyme-linked immunosorbent assay (IC ELISA), immunohistochemistry (IHC), and AGAR gel test. Whereas most of the confirmatory diagnostic procedures require pathological samples to be stored frozen to preserve integrity of the peste des petits ruminants (PPR) virus RNA, samples for IHC tests are preserved in 10 % formalin. In this study, nine formalin-fixed pathological samples from three goats suspected of PPR were processed for extraction of PPR viral RNA and analyzed for detection with real-time reverse transcription–polymerase chain reaction (qRT-PCR) assay. The results showed that five out of the nine tested samples returned positive for presences PPR viral genome. This study has established that field pathological samples of PPR-suspected cases, collected and stored in 10 % formalin for up 2 years, could be used for PPR virus RNA extraction for disease virus confirmation. Keywords Peste des petits ruminants virus . Ribonucleic acid . Formalin-fixed tissues S. M. Kihu (*) : G. C. Gitao : L. C. Bebora : M. J. Njenga : N. Maingi : R. G. Wahome Faculty of Veterinary Medicine, University of Nairobi, P.O. Box 29053, 00625 Uthiru, Kenya e-mail: [email protected] G. G. Wairire Faculty of Arts, University of Nairobi, P.O. Box 30197, 00100 Nairobi, Kenya J. O. Oyugi : E. Lutomia University of Nairobi Institute of Tropical and Infectious Diseases (UNITID), P.O. Box 19676, 00202 Nairobi, Kenya
Introduction Peste des petits ruminants (PPR) is a highly infectious and often fatal disease of sheep, goats, and wild small ruminants that is caused by peste des petits ruminants virus (PPRV) classified under genus Morbillivirus in the family Paramyxoviridae (Libeau et al. 2014). PPR disease outbreaks can cause mortality as high as 90 % in immunologically naive sheep and goat populations resulting in significant negative impact to food security and pastoral economies of subsistence farmers who are dependent on small ruminants particularly in the developing countries of the tropical region (Libeau et al. 2014). Peste des petits ruminants is currently confined to Middle East, South and central Asia, China, and Africa regions that host most of the developing countries (Munir et al. 2013). Effective diagnosis and eventual control of PPR is thus considered an essential element in the fight for global food security and poverty alleviation in these developing countries (Dundon et al. 2014). Tentative diagnosis of PPR is done through observation of clinical disease and serology, whereas confirmatory diagnosis of PPR is done through various diagnostic techniques among them laboratory isolation of PPR virus in cell culture, detection of viral antigen by immunocapture enzyme-linked immunosorbent assay (IC ELISA), immunohistochemistry (IHC), and AGAR gel test, and molecular detection of PPR viral RNA using real-time reverse transcription–polymerase chain reaction (qRT-PCR) assay (Troung et al. 2014; Munir et al. 2012; Aytekin et al. 2011; Adombi et al. 2011). Apart from IHC, the other confirmatory diagnostic procedures require pathological samples to be stored frozen to preserve the integrity of the PPR virus RNA thus yielding high-quality RNA during the extraction process. In situations where samples are preserved as formalin-fixed tissues and are to be used in extraction of viral RNA, the quality and quantity of RNA extracted is dependent on the initial process of fixation and
248
Trop Anim Health Prod (2015) 47:247–249
the extent of reversal of crosslinks formed by proteins and nucleic acids involving hydroxymethylene bridges of formalin that confound extraction of intact RNA from formalinfixed tissues (Scicchitano et al. 2006). A comparative study of methods for RNA extraction from formalin-fixed tissues reported by Sharma et al. (2012) concluded that formalinfixed tissues can be used for RNA extraction to be used for gene expression, pathogen detection, and epidemiological studies. In this study, an attempt is made to extract and detect PPR viral RNA from formalin-fixed tissues collected from field cases of PPR disease.
Materials and methods The study samples were collected from Lotakaa village herd in Turkana, Kenya (N 03° 38 390; S 034° 50 987) in 2011. Nine laboratory samples that included lung tissues and mesenteric and mediastinal lymph nodes; and colons were collected from carcasses and preserved in 10 % formalin. Extraction of PPR RNA was as outlined by RNeasy® FFPE kit protocol supplied by QIAGEN®. The extracted RNA samples were analyzed in a MicroAmp® Optical 96-well reaction plate by Abiprism® 7500 (Applied Biosystems) thermocycler using a specific qRT-PCR assay called TaqVet™ Peste des Petits Ruminants Virus kit which had a set of primers/probes designed from the Nterminus of the N-gene in the Mix PPRV based on a protocol supplied by manufacturer. The forward primer matched positions 483>508 in the (5-AGAGTTCAATATGTTRTTAGCC TCCAT-3); the TaqMan® probe positions 551>576 (FAM-5CACCGGAYACKGCAGCTGACTCAGAA-3- MGB) and the reverse primer were located at positions 603
