Veterinary Microbiology 168 (2014) 197–201
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Detection and isolation of 2009 pandemic influenza A/H1N1 virus in commercial piggery, Lagos Nigeria C.A. Meseko a,b,*, G.N. Odaibo b, D.O. Olaleye b a b
Virology Department, College of Medicine, University of Ibadan, Ibadan, Nigeria Regional Center for Animal Influenza, National Veterinary Research Institute, Vom, Nigeria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 May 2013 Received in revised form 19 September 2013 Accepted 4 November 2013
WHO declared pandemic of A/H1N1influenza in 2009 following global spread of the newly emerged strain of the virus from swine. Presently there is a dearth of data on the ecology of pandemic influenza H1N1 required for planning of intervention measures in sub Saharan Africa. Herein we report isolation of 2009 pandemic influenza A/H1N1 in an intensive mega piggery farms operation in South West Nigeria. Sentinel surveillance was carried out in a cohort of intensively reared pigs over a period of two years. Nasal swab specimens were collected at monthly interval from observed clinical cases of influenza like illness in pigs and pig handlers. Samples were analyzed by real time RT-PCR and isolation in chicken embryonated eggs. A total of 227 clinical cases of influenza like illness were observed among pigs out of which 31 (13.7%) were positive for influenza A matrix gene by real time RT-PCR. Virus isolation yielded 29 (12%) isolates out of which 18 (18%) were identified as influenza A/H1N1 by Heamaglutination Inhibition test using H1 antisera. RT-PCR positive samples were subtyped as 2009 pandemic A/H1N1 with subtype specific primers and probes. This is the first report of detection and isolation of pandemic influenza H1N1 from pigs in Nigeria. Continuous circulation of this virus in pigs may cause reassortments with seasonal influenza or mutations and substitutions in the gene that may result in the emergence of novel or pandemic influenza virus of economic and public health importance. Nigeria is considered a geographical hotspot of zoonotic diseases, which necessitate active surveillance and monitoring of emerging pandemic threats. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Pandemic influenza Detection Isolation Pigs Nigeria
1. Introduction The major pandemic of 1918, named the Spanish flu was hypothesized to have originated from avian sources and was transmitted from human to pigs in the course of the pandemic (Reid et al., 1999). This virus was maintained in pig population as classical swine influenza,
* Corresponding author at: Virology Department, College of Medicine, University of Ibadan, Ibadan, Nigeria. Tel.: +234 8039183988. E-mail addresses: [email protected], [email protected] (C.A. Meseko), [email protected] (G.N. Odaibo), [email protected] (D.O. Olaleye). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.11.003
occasionally causing epidemics and pandemics in human contacts (Dowdle, 1997; Myers et al., 2007). Pigs are particularly important in epidemiology of influenza virus because of their susceptibility to both avian and human strains of the virus (Ma et al., 2008). Many subtypes of influenza virus have thus emerged in recent past as a result of re-assortments and mutations of influenza virus circulating in animal reservoirs particularly avian and swine (Alexander and Brown, 2000). The major subtypes of classical swine influenza virus circulating in nature include variants of H1N1, H1N2 and H3N2, many of which can be detected at the human–animal interface (Brown, 2000; Yu et al., 2009). Lack of consistent surveillance in animal reservoir led to failure to detect novel reassortants
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C.A. Meseko et al. / Veterinary Microbiology 168 (2014) 197–201
that eventually emerged as pandemics in human. WHO declared the first pandemic of the 21st century in June 2009 after swine origin influenza A/H1N1 subtype that was first reported in Mexico, spread through 5 continents including Africa. The H1N1 strain has not circulated previously in humans and is entirely new (WHO, 2009a). Shortly after the outbreak in human host, infections were reported in pigs in North America, Europe and Asia apparently transmitted from human to pigs (Garten et al., 2009). On the other hand, virological data on the detection and isolation of swine influenza in Africa are limited due to limited surveillance activities in the region. Yet knowledge of the circulation of influenza in pigs and other domestic animals in this region is equally important for global influenza pandemic preparedness and control. This study was therefore designed to detect and isolate influenza virus that may be circulating at the human– animal interface in a large commercial pig farm settlement in south western Nigeria. 2. Materials and method 2.1. Study population
corrugated zinc. Each farm houses an average of 200 pigs with separate rooms for breeders, wean piglets and growers. The farms are closely located with less than five meters between each farmhouse. Fattened pigs are regularly sold to pork meat vendors who slaughter the animals at any of the three slaughter slabs provided in the estate and pigs are also transported beyond the estate to other parts of the country and across the border to neighbouring West African countries. Strict clinical cases of influenza-like illness based on case definition of fever, cough and respiratory distress in pigs and pig handlers were monitored for two years (July 2010–June 2012). Nasal swab specimens were carefully collected into virus transport medium. Nasal swabs were collected from pig handlers with clinical symptoms of influenza-like illness (ILI) or history or ILI less than ten days. Pig handlers who consented were also bled and blood samples were also collected from pigs. Samples were kept on ice and transported in cold insulated box at about 4 8C to the laboratory where the tubes were held at 4 8C before analysis or stored at 80 8C when they were to be held for longer than 24 h before analysis. 2.2. Nucleic acid extraction
Sentinel surveillance involving continuous monitoring of a population cluster at monthly interval was carried out to determine the activity of infection with swine influenza virus at the human–animal interface in a cohort of intensively reared, peri-urban pig estate in Lagos Nigeria. The farm is situated in Latitude: 6.413207 N and Longitude: 3.193127 E (Fig. 1). There are about 800,000 pigs owned by about 5000 registered farmers with 2000 farm service providers within an area covering 44 ha of land. Pigs are kept in cement block pens with concrete floors provided with drains and roofed with asbestos or
Nucleic acid was extracted from swab fluid after centrifugation at 1500–2000 rpm for 15–30 min to obtain clear supernatant. Qiagen RNAeasy commercial kit (Qiagen, Hilden Germany) was used for RNA extraction according to manufacturer’s instruction. Briefly; reagents and buffers were equilibrated to room temperature (15– 25 8C) and wash buffers reconstituted accordingly. About 140 ml of swab supernatant in VTM was added to Buffer AVL – carrier RNA in the micro-centrifuge tube with extract control tubes. The tubes were mix by pulse vortex
Fig. 1. Map of Nigeria-study area.
C.A. Meseko et al. / Veterinary Microbiology 168 (2014) 197–201
for 15 s and incubated at room temperature for 10 min and briefly centrifuged to remove drops from inside the lid. 560 ml of absolute ethanol was added to samples and mixed by pulse vortexing for 15 s. 630 ml of the solution was thereafter pipette into the QIAamp minispin column in a 2 ml collection tube and centrifuged at 8000 rpm, for 1 min. The QIAamp minispin column was transferred into 2 ml collection tube for washing steps. The first washing step involved dispensing 500 ml of buffer AW1 into the minispin column and centrifuged at 8000 rpm for 1 min rate. 500 ml of AW2 was also dispensed into the minispin column and centrifuged at 14,000 rpm for 3 min for second washing step. The minispin column was thereafter placed in a clean, sterile, RNAse free 1.5 ml microcentrifuge tube and Nucleic acid eluted by dispensing 60 ml of RNA-free water and centrifuged at 8000 rpm for 1 min. The elute containing viral RNA was either used immediately to run RT-PCR or stored at 20 8C until tested. 2.3. Real time RT-PCR Extracted nucleic acid was analyzed for influenza A matrix gene by one step quantitative PCR (qRT-PCR) for the detection and quantification of RNA using real-time detection protocol on Applied Biosystem platform. The system combines superscript III ReverseTranscriptase (RT) and platinum TaqDNA Polymerase in a single enzyme mix: both cDNA synthesis and PCR are performed in a single reaction. Influenza virus gene specific primers and probes designed for generic matrix gene (F: 50 -AGATGAGTCTTCTAACCGAGGTCG-30 R: 30 -TGCAAAGACACTTTCCAGTCTCTG-50 , FAM-50 -TCAGGCCCCCTCAAAGCCGA-30 ) was used for nucleotide amplification. Cocktail of master mix was prepared by dispensing 5.5 ml nuclease free water, 0.5 ml of forward, reverse primers and probe, 0.5 ml superscript tag and 12.5 ml 2 PCR master mix enzyme for a single reaction multiplied by the number of samples tested. Cycling condition was set at: Reverse transcription 42–50 8C for 30 min, RT inactivation/Taq activation 95 8C 2–15 min, PCR amplification (35–45 cycles), Denaturation/ primer annealing/Template extension at 95 8C, 45–60 8C at 15 s and 5–60 s respectively. Subtype identification with primers and probes designed for the Haemaglutination (HA) gene were used viz.: swine influenza A (F: GCA CGG TCA GCA CTT ATY CTR AG, R: GTG RGC TGG GTT TTC ATT TGG TC, P: CYA CTG CAA GCC CAT ACA CAC AAG CAG GCA), pandemic H1, (F: GTG CTA TAA ACA CCA GCC TYC CA, R: CGG GAT ATT CCT TAA TCC TGT RGC, P: CA GAA TAT ACA ‘‘T’’CC RGT CAC AAT TGG ARA A). Primers, probes were sourced from CDC using the WHO/CDC pandemic influenza A/H1N1 protocol.
199
observed daily by candling for embryo vitality. Dead egg or eggs that were terminated after 5 days post-inoculation were chilled at +48 C or overnight before harvesting. Alternatively eggs were rapidly chilled at 20 8C for 30 min. Samples without heamaglutinating activity were harvested for passage in another set of eggs. Two passages were carried out before considering a sample negative for influenza virus isolation. 2.5. Haemaglutination and Haemaglutination Inhibition Rapid Haemaglutination (HA) was performed as spot test on a flat white surface using 10% suspension of chicken red blood cell (RBC). Positive heamaglutinating agents were further tested quantitatively on Microtitre plate and those with high HA (>23) titers were harvested and stored in aliquots as viral isolate at 80 8C. Haemaglutination Inhibition (HI) of unknown influenza A virus harvest was identified according to WHO protocol (WHO, 2009b) using antiserum and reference antigen provided in the 2011 WHO/CDC Influenza surveillance kit. A sample is considered positive for a specific H-subtype if haemagglutination of chicken red blood cell (RBC) is inhibited. The test is considered valid if the positive reference antigen and its homologous antiserum demonstrate the expected HI titer and the back titration of each antigen (unknown and positive control) is 4 HAUs/25 ml. 3. Results Two hundred and twenty seven clinical cases of influenza-like illness were observed. One hundred and twenty seven (55.94%) samples were collected from female animals and 98 (43.17%) from males while 2 (0.88%) of the samples were from castrated male. Greater proportion (64.3%) of grower pigs had severe cough, followed by adult pigs (20.26%), weaners (13.65%) and piglets (1.76%). The highest number of cases and samples collected were in the cold windy harmattan months of Novemeber to Janauary (Fig. 2), subsequently more isolates were obtained during this period, thirty one (13.7%) out of the 227 specimens collected from July 2010 and June 2012 were positive for influenza A by real time RT-PCR. Twenty nine out of the original 227 specimens (12.8%) were positive for virus isolation and 18 out of 29 (7.9%) isolates were identified as 2009 pandemic influenza. Further identification of the remaining 11 isolates is in progress.
2.4. Virus isolation Aliquots of nasal swab samples that were positive by RT-PCR were inoculated in 8–10 day old specific antibody negative chicken embryonated eggs sourced from local hatchery. Holes were drilled through the shell above the air space of viable eggs and 200 ml of inoculums injected into the allantoic cavity with 1 ml syringe. Eggs were sealed and incubated at 358 C in a humidify incubator and
Fig. 2. Monthly distribution of number of cases and number positive.
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4. Discussion This report is the first evidence of circulation of 2009 pandemic H1N1 influenza virus in pigs in Nigeria and the second in Africa. Earlier observation of human pandemic influenza virus transmitted to pigs was reported in Canada (Pereda et al., 2010). Zoonotic transmission episodes often occur where pigs are intensively raised (Myers et al., 2007). Recently, isolated case of 2009 pandemic influenza H1N1 was detected in a free range pig in Cameroon but there was no evidence of further circulation or transmission (Njabo et al., 2012). In this study, clinical signs related to swine influenza in 227 cases were observed within a geographical area with widespread infection among farms in the estate. Virus detection and isolation was continuous throughout the two-year period of study with higher isolation rate during the harmattan months of November to January (Fig. 2). However, there were no clinical or laboratory confirmed cases of influenza by virus isolation among human handlers of pigs including pen attendants, farm owners, butchers and traders in live or slaughtered pigs. However, evidence of subclinical human infection by detection of antibodies to pandemic H1N1 (reported separately) was observed. Prior to this investigation, human cases of pandemic A/H1N1 influenza were recorded in the study area. Apparently the 2009 pandemic influenza from pigs in the farm estate may have been transmitted from human because there has not been any previously reported case of either classical or pandemic influenza in swine in the study area but human cases of pandemic H1N1 in human (Dalhatu et al., 2012) It is worthy of note that the entire virus that was conclusively identified was confirmed by both molecular and serological tests to be 2009 pandemic influenza H1N1. Serological cross reactivity is however possible between classical and pandemic H1N1 but subtype specific primers and probe designed for pandemic H1N1 was used in this analysis. No classical or other strain of swine influenza was detectable or isolated because sampling was strictly based on clinical case definition of fever, cough and acute respiratory distress (Yu et al., 2009). Pigs showing this signs were prostrate, were off feed and some were emaciated but only 2 (
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Detection and isolation of 2009 pandemic influenza A/H1N1 virus in commercial piggery, Lagos Nigeria C.A. Meseko a,b,*, G.N. Odaibo b, D.O. Olaleye b a b
Virology Department, College of Medicine, University of Ibadan, Ibadan, Nigeria Regional Center for Animal Influenza, National Veterinary Research Institute, Vom, Nigeria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 May 2013 Received in revised form 19 September 2013 Accepted 4 November 2013
WHO declared pandemic of A/H1N1influenza in 2009 following global spread of the newly emerged strain of the virus from swine. Presently there is a dearth of data on the ecology of pandemic influenza H1N1 required for planning of intervention measures in sub Saharan Africa. Herein we report isolation of 2009 pandemic influenza A/H1N1 in an intensive mega piggery farms operation in South West Nigeria. Sentinel surveillance was carried out in a cohort of intensively reared pigs over a period of two years. Nasal swab specimens were collected at monthly interval from observed clinical cases of influenza like illness in pigs and pig handlers. Samples were analyzed by real time RT-PCR and isolation in chicken embryonated eggs. A total of 227 clinical cases of influenza like illness were observed among pigs out of which 31 (13.7%) were positive for influenza A matrix gene by real time RT-PCR. Virus isolation yielded 29 (12%) isolates out of which 18 (18%) were identified as influenza A/H1N1 by Heamaglutination Inhibition test using H1 antisera. RT-PCR positive samples were subtyped as 2009 pandemic A/H1N1 with subtype specific primers and probes. This is the first report of detection and isolation of pandemic influenza H1N1 from pigs in Nigeria. Continuous circulation of this virus in pigs may cause reassortments with seasonal influenza or mutations and substitutions in the gene that may result in the emergence of novel or pandemic influenza virus of economic and public health importance. Nigeria is considered a geographical hotspot of zoonotic diseases, which necessitate active surveillance and monitoring of emerging pandemic threats. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Pandemic influenza Detection Isolation Pigs Nigeria
1. Introduction The major pandemic of 1918, named the Spanish flu was hypothesized to have originated from avian sources and was transmitted from human to pigs in the course of the pandemic (Reid et al., 1999). This virus was maintained in pig population as classical swine influenza,
* Corresponding author at: Virology Department, College of Medicine, University of Ibadan, Ibadan, Nigeria. Tel.: +234 8039183988. E-mail addresses: [email protected], [email protected] (C.A. Meseko), [email protected] (G.N. Odaibo), [email protected] (D.O. Olaleye). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.11.003
occasionally causing epidemics and pandemics in human contacts (Dowdle, 1997; Myers et al., 2007). Pigs are particularly important in epidemiology of influenza virus because of their susceptibility to both avian and human strains of the virus (Ma et al., 2008). Many subtypes of influenza virus have thus emerged in recent past as a result of re-assortments and mutations of influenza virus circulating in animal reservoirs particularly avian and swine (Alexander and Brown, 2000). The major subtypes of classical swine influenza virus circulating in nature include variants of H1N1, H1N2 and H3N2, many of which can be detected at the human–animal interface (Brown, 2000; Yu et al., 2009). Lack of consistent surveillance in animal reservoir led to failure to detect novel reassortants
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C.A. Meseko et al. / Veterinary Microbiology 168 (2014) 197–201
that eventually emerged as pandemics in human. WHO declared the first pandemic of the 21st century in June 2009 after swine origin influenza A/H1N1 subtype that was first reported in Mexico, spread through 5 continents including Africa. The H1N1 strain has not circulated previously in humans and is entirely new (WHO, 2009a). Shortly after the outbreak in human host, infections were reported in pigs in North America, Europe and Asia apparently transmitted from human to pigs (Garten et al., 2009). On the other hand, virological data on the detection and isolation of swine influenza in Africa are limited due to limited surveillance activities in the region. Yet knowledge of the circulation of influenza in pigs and other domestic animals in this region is equally important for global influenza pandemic preparedness and control. This study was therefore designed to detect and isolate influenza virus that may be circulating at the human– animal interface in a large commercial pig farm settlement in south western Nigeria. 2. Materials and method 2.1. Study population
corrugated zinc. Each farm houses an average of 200 pigs with separate rooms for breeders, wean piglets and growers. The farms are closely located with less than five meters between each farmhouse. Fattened pigs are regularly sold to pork meat vendors who slaughter the animals at any of the three slaughter slabs provided in the estate and pigs are also transported beyond the estate to other parts of the country and across the border to neighbouring West African countries. Strict clinical cases of influenza-like illness based on case definition of fever, cough and respiratory distress in pigs and pig handlers were monitored for two years (July 2010–June 2012). Nasal swab specimens were carefully collected into virus transport medium. Nasal swabs were collected from pig handlers with clinical symptoms of influenza-like illness (ILI) or history or ILI less than ten days. Pig handlers who consented were also bled and blood samples were also collected from pigs. Samples were kept on ice and transported in cold insulated box at about 4 8C to the laboratory where the tubes were held at 4 8C before analysis or stored at 80 8C when they were to be held for longer than 24 h before analysis. 2.2. Nucleic acid extraction
Sentinel surveillance involving continuous monitoring of a population cluster at monthly interval was carried out to determine the activity of infection with swine influenza virus at the human–animal interface in a cohort of intensively reared, peri-urban pig estate in Lagos Nigeria. The farm is situated in Latitude: 6.413207 N and Longitude: 3.193127 E (Fig. 1). There are about 800,000 pigs owned by about 5000 registered farmers with 2000 farm service providers within an area covering 44 ha of land. Pigs are kept in cement block pens with concrete floors provided with drains and roofed with asbestos or
Nucleic acid was extracted from swab fluid after centrifugation at 1500–2000 rpm for 15–30 min to obtain clear supernatant. Qiagen RNAeasy commercial kit (Qiagen, Hilden Germany) was used for RNA extraction according to manufacturer’s instruction. Briefly; reagents and buffers were equilibrated to room temperature (15– 25 8C) and wash buffers reconstituted accordingly. About 140 ml of swab supernatant in VTM was added to Buffer AVL – carrier RNA in the micro-centrifuge tube with extract control tubes. The tubes were mix by pulse vortex
Fig. 1. Map of Nigeria-study area.
C.A. Meseko et al. / Veterinary Microbiology 168 (2014) 197–201
for 15 s and incubated at room temperature for 10 min and briefly centrifuged to remove drops from inside the lid. 560 ml of absolute ethanol was added to samples and mixed by pulse vortexing for 15 s. 630 ml of the solution was thereafter pipette into the QIAamp minispin column in a 2 ml collection tube and centrifuged at 8000 rpm, for 1 min. The QIAamp minispin column was transferred into 2 ml collection tube for washing steps. The first washing step involved dispensing 500 ml of buffer AW1 into the minispin column and centrifuged at 8000 rpm for 1 min rate. 500 ml of AW2 was also dispensed into the minispin column and centrifuged at 14,000 rpm for 3 min for second washing step. The minispin column was thereafter placed in a clean, sterile, RNAse free 1.5 ml microcentrifuge tube and Nucleic acid eluted by dispensing 60 ml of RNA-free water and centrifuged at 8000 rpm for 1 min. The elute containing viral RNA was either used immediately to run RT-PCR or stored at 20 8C until tested. 2.3. Real time RT-PCR Extracted nucleic acid was analyzed for influenza A matrix gene by one step quantitative PCR (qRT-PCR) for the detection and quantification of RNA using real-time detection protocol on Applied Biosystem platform. The system combines superscript III ReverseTranscriptase (RT) and platinum TaqDNA Polymerase in a single enzyme mix: both cDNA synthesis and PCR are performed in a single reaction. Influenza virus gene specific primers and probes designed for generic matrix gene (F: 50 -AGATGAGTCTTCTAACCGAGGTCG-30 R: 30 -TGCAAAGACACTTTCCAGTCTCTG-50 , FAM-50 -TCAGGCCCCCTCAAAGCCGA-30 ) was used for nucleotide amplification. Cocktail of master mix was prepared by dispensing 5.5 ml nuclease free water, 0.5 ml of forward, reverse primers and probe, 0.5 ml superscript tag and 12.5 ml 2 PCR master mix enzyme for a single reaction multiplied by the number of samples tested. Cycling condition was set at: Reverse transcription 42–50 8C for 30 min, RT inactivation/Taq activation 95 8C 2–15 min, PCR amplification (35–45 cycles), Denaturation/ primer annealing/Template extension at 95 8C, 45–60 8C at 15 s and 5–60 s respectively. Subtype identification with primers and probes designed for the Haemaglutination (HA) gene were used viz.: swine influenza A (F: GCA CGG TCA GCA CTT ATY CTR AG, R: GTG RGC TGG GTT TTC ATT TGG TC, P: CYA CTG CAA GCC CAT ACA CAC AAG CAG GCA), pandemic H1, (F: GTG CTA TAA ACA CCA GCC TYC CA, R: CGG GAT ATT CCT TAA TCC TGT RGC, P: CA GAA TAT ACA ‘‘T’’CC RGT CAC AAT TGG ARA A). Primers, probes were sourced from CDC using the WHO/CDC pandemic influenza A/H1N1 protocol.
199
observed daily by candling for embryo vitality. Dead egg or eggs that were terminated after 5 days post-inoculation were chilled at +48 C or overnight before harvesting. Alternatively eggs were rapidly chilled at 20 8C for 30 min. Samples without heamaglutinating activity were harvested for passage in another set of eggs. Two passages were carried out before considering a sample negative for influenza virus isolation. 2.5. Haemaglutination and Haemaglutination Inhibition Rapid Haemaglutination (HA) was performed as spot test on a flat white surface using 10% suspension of chicken red blood cell (RBC). Positive heamaglutinating agents were further tested quantitatively on Microtitre plate and those with high HA (>23) titers were harvested and stored in aliquots as viral isolate at 80 8C. Haemaglutination Inhibition (HI) of unknown influenza A virus harvest was identified according to WHO protocol (WHO, 2009b) using antiserum and reference antigen provided in the 2011 WHO/CDC Influenza surveillance kit. A sample is considered positive for a specific H-subtype if haemagglutination of chicken red blood cell (RBC) is inhibited. The test is considered valid if the positive reference antigen and its homologous antiserum demonstrate the expected HI titer and the back titration of each antigen (unknown and positive control) is 4 HAUs/25 ml. 3. Results Two hundred and twenty seven clinical cases of influenza-like illness were observed. One hundred and twenty seven (55.94%) samples were collected from female animals and 98 (43.17%) from males while 2 (0.88%) of the samples were from castrated male. Greater proportion (64.3%) of grower pigs had severe cough, followed by adult pigs (20.26%), weaners (13.65%) and piglets (1.76%). The highest number of cases and samples collected were in the cold windy harmattan months of Novemeber to Janauary (Fig. 2), subsequently more isolates were obtained during this period, thirty one (13.7%) out of the 227 specimens collected from July 2010 and June 2012 were positive for influenza A by real time RT-PCR. Twenty nine out of the original 227 specimens (12.8%) were positive for virus isolation and 18 out of 29 (7.9%) isolates were identified as 2009 pandemic influenza. Further identification of the remaining 11 isolates is in progress.
2.4. Virus isolation Aliquots of nasal swab samples that were positive by RT-PCR were inoculated in 8–10 day old specific antibody negative chicken embryonated eggs sourced from local hatchery. Holes were drilled through the shell above the air space of viable eggs and 200 ml of inoculums injected into the allantoic cavity with 1 ml syringe. Eggs were sealed and incubated at 358 C in a humidify incubator and
Fig. 2. Monthly distribution of number of cases and number positive.
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4. Discussion This report is the first evidence of circulation of 2009 pandemic H1N1 influenza virus in pigs in Nigeria and the second in Africa. Earlier observation of human pandemic influenza virus transmitted to pigs was reported in Canada (Pereda et al., 2010). Zoonotic transmission episodes often occur where pigs are intensively raised (Myers et al., 2007). Recently, isolated case of 2009 pandemic influenza H1N1 was detected in a free range pig in Cameroon but there was no evidence of further circulation or transmission (Njabo et al., 2012). In this study, clinical signs related to swine influenza in 227 cases were observed within a geographical area with widespread infection among farms in the estate. Virus detection and isolation was continuous throughout the two-year period of study with higher isolation rate during the harmattan months of November to January (Fig. 2). However, there were no clinical or laboratory confirmed cases of influenza by virus isolation among human handlers of pigs including pen attendants, farm owners, butchers and traders in live or slaughtered pigs. However, evidence of subclinical human infection by detection of antibodies to pandemic H1N1 (reported separately) was observed. Prior to this investigation, human cases of pandemic A/H1N1 influenza were recorded in the study area. Apparently the 2009 pandemic influenza from pigs in the farm estate may have been transmitted from human because there has not been any previously reported case of either classical or pandemic influenza in swine in the study area but human cases of pandemic H1N1 in human (Dalhatu et al., 2012) It is worthy of note that the entire virus that was conclusively identified was confirmed by both molecular and serological tests to be 2009 pandemic influenza H1N1. Serological cross reactivity is however possible between classical and pandemic H1N1 but subtype specific primers and probe designed for pandemic H1N1 was used in this analysis. No classical or other strain of swine influenza was detectable or isolated because sampling was strictly based on clinical case definition of fever, cough and acute respiratory distress (Yu et al., 2009). Pigs showing this signs were prostrate, were off feed and some were emaciated but only 2 (
