2009 influenza A virus in Burkina Faso

Journal Pre-proof Serological evidence of swine exposure to pandemic H1N1/2009 influenza A virus in Burkina Faso ´ Sausy, Assana Cisse, ´ Tani Sagna, Abdoul Dieudonne´ Tialla, Aurelie ´ Kader Ilboudo, Georges Anicet Ouedraogo, Judith M. Hubschen, ¨ ´ Zekiba Tarnagda, Chantal J. Snoeck

PII:

S0378-1135(19)30975-7

DOI:

https://doi.org/10.1016/j.vetmic.2019.108572

Reference:

VETMIC 108572

To appear in:

Veterinary Microbiology

Received Date:

14 August 2019

Revised Date:

29 December 2019

Accepted Date:

30 December 2019

´ Please cite this article as: Tialla D, Sausy A, Cisse´ A, Sagna T, Ilboudo AK, Ouedraogo GA, Hubschen ¨ JM, Tarnagda Z, Snoeck CJ, Serological evidence of swine exposure to pandemic H1N1/2009 influenza A virus in Burkina Faso, Veterinary Microbiology (2019), doi: https://doi.org/10.1016/j.vetmic.2019.108572

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Serological evidence of swine exposure to pandemic H1N1/2009 influenza A virus in Burkina Faso

Tialla Dieudonnéa,b, Sausy Auréliec, Cissé Assanaa, Sagna Tania, Ilboudo Abdoul Kadera, Ouédraogo Georges Anicetd, Hübschen Judith M.c, Tarnagda Zékibaa, Snoeck Chantal J.c*

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[email protected]

Unité des Maladies à potentiel Epidémique, Maladies Emergentes et Zoonoses (UMEMEZ),

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Département Biomédical et Santé Publique, Institut de Recherche en Sciences de la Santé (IRSS), 399, Avenue de la Liberté 01, BP 545, Bobo-Dioulasso, Burkina Faso. [email protected];

Ecole Nationale de l’Elevage et de la Santé Animale (ENESA), Secteur 28, Ouagadougou, Burkina

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b

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[email protected]; [email protected]; [email protected]; [email protected]

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Infectious Diseases Research Unit, Department of Infection and Immunity, Luxembourg Institute

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of Health (LIH), 29 rue Henri Koch, L-4354 Esch-sur-Alzette, Luxembourg. [email protected]; [email protected]; [email protected] Laboratoire de Recherche et d’Enseignement en Santé et Biotechnologies Animales (LARESBA),

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Université Nazi Boni, 01 BP 109, Bobo-Dioulasso, Burkina Faso. [email protected]

*

Corresponding author: Chantal J. Snoeck, Department of Infection and Immunity, Luxembourg

Institute of Health, 29 rue Henri Koch, L-4354 Esch-sur-Alzette, Luxembourg. Tel: +352 26970627; Fax: +352 26970660.

Highlights Pigs were exposed to pandemic H1N1/2009 influenza A virus in Burkina Faso

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Cross-reactivity against other swine H1N1 strains was observed

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Reverse zoonosis strongly influences influenza virus ecology in pigs in Africa

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Farmers are likely key players in driving human-to-swine virus transmission

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Abstract

Despite improvement of human and avian influenza surveillance, swine influenza surveillance in

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sub-Saharan Africa is scarce and pandemic preparedness is still deemed inadequate, including in Burkina Faso. This cross-sectional study therefore aimed to investigate the (past) exposure of pigs to influenza A viruses. Practices of people with occupational contacts with pigs and their knowledge on influenza A were investigated in order to formulate future prevention guidelines. In

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2016-2017, pig nasopharyngeal swabs and sera were collected and screened for the presence of influenza virus by RT-PCR or of anti-influenza antibodies by competitive ELISA. Seropositive samples were further characterized in virus microneutralization assays against human and swine H1N1 virus strains. Nasopharyngeal swabs were obtained from people with occupational contact with pigs and screened similarly. Demographic data as well as practices related to their profession were recorded. No influenza A virus was detected in nasopharyngeal swabs in humans (n=358) or

in pigs (n=600). Seroprevalence in pigs reached 6.8% (41/600) and seropositive animals were found in 50.0% of extensive settings (10/20) and 19.0% of (semi-)intensive farms (4/21). All positive sera reacted against the pandemic H1N1/2009 strain, while seropositivity against two Eurasian avianlike and one American swine H1N1 strains and individual titers were lower. These results suggested a human-to-swine transmission of pandemic H1N1/2009 virus and cross-reactivity to other H1N1 strains. Farmers with higher frequency of contact to pigs, absence of protective equipment and lack of knowledge on zoonoses are likely key players in driving human-to-swine virus transmission.

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Keywords : Epidemiology, Influenza A virus, pandemic H1N1/2009, Pigs, Public Health, Burkina

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Faso, Reverse zoonosis.

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Introduction

The risk for the emergence of a pandemic influenza A virus is multifactorial, including virus

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diversity in its hosts, animal densities and intermingling, frequency of animal-to-human contacts, human population density as well as the virus properties (Trock et al., 2015). Therefore, the emergence threat varies across the globe (Berger et al., 2018) and can only be properly estimated

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based on continuous surveillance.

Although the swine influenza virus strains circulating in North America, Europe and Asia are relatively well described, our knowledge on swine influenza viruses in sub-Saharan Africa is still scarce. Despite previous epidemics (e.g. A/H5N1), pandemics (e.g. pandemic H1N1/2009) and continuous improvement of national surveillance systems, preparedness for pandemic influenza in sub-Saharan Africa is still considered inadequate (Sambala et al., 2018). West Africa should

however be regarded as a priority region for influenza surveillance, due to the high potential for animal-to-human virus transmission and secondary human-to-human spread (Berger et al., 2018). Furthermore, surveillance in pigs is notably weak in West Africa (Meseko et al., 2014), despite the role of pigs in influenza ecology and the increasing popularity of pig husbandry in the region. People with (occupational) exposure to pigs are at higher risk of contracting swine influenza viruses (Ma et al., 2015). Cases of swine influenza viruses are periodically reported, especially in the USA, where the largest outbreak took place in 2011-2012, with many patients with swine contact at

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agricultural fairs (Greenbaum et al., 2015). Conversely, human-to-swine virus transmission, a

phenomenon known as reverse zoonosis, is frequent and substantially influences influenza virus epidemiology and diversity in swine (Rajao et al., 2018).

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In Burkina Faso, sentinel surveillance in poultry and in human was initiated in 2006 and 2010

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respectively (Ducatez et al., 2007; Tarnagda et al., 2014), while surveillance in swine is virtually nonexistent. This study therefore aimed at investigating the current exposure of pigs and people

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with occupational contacts with pigs as well as the past exposure of pigs to influenza A viruses to assess the extent of virus circulation. The study also aimed at understanding practices of people

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with exposure to swine and their knowledge on influenza A virus in order to formulate future prevention guidelines.

Material and Methods

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Ethical approval

This study received ethical clearance from Centre Muraz ethical committee (number 201615/MS/SG/CM/IEC). Prior to sample collection, the aim of the study was explained to all participants and pig owners and signed informed consents were obtained.

Study population and data collection Veterinary services reported a total of 62 pigs farms in the vicinity of Bobo-Dioulasso city in the west part of Burkina Faso, of which 41 were randomly selected. In October-December 2016 and in June-July 2017, a total of 600 nasopharyngeal swabs and sera were collected in 21 (semi-) intensive farms (herd sizes: 44-504), in 20 extensive farms (herd sizes: 11-36) and in one slaughterhouse (Table 1). Two visits were made to each farm. The first visit aimed to sensitize farmers about the study and the second was focused on sample collection. A questionnaire was administered to

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farmers in French or local language to collect data on the husbandry system, pig herd size, the

presence of animal species on the farm. The age, sex, breed of each pig sampled were also recorded. On all farms surveyed, poultry were present in various flock sizes (Table 1).

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During the same periods, nasopharyngeal swabs were obtained from people with occupational

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contact with pigs (farmers, n=73; veterinarians, n=99; slaughterhouse workers, n=100, gardeners, n=86). Demographic data from all study participants (n=358), profession, type and frequency of

practices and awareness survey.

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Sample collection

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contact with pigs as well as the use of personal protective equipment were recorded through a brief

Nasopharyngeal swabs were collected from both humans and pigs using a sterile flocked nylon swab (Copan/MLS, Menen, Belgium), then placed directly in a tube containing 3 mL of viral

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transport medium (VTM). Blood samples were taken from the jugular vein of pigs in a dry tube with a sterile needle. All samples were immediately placed in a cooler and transported to the laboratory in the evening. Serum was allowed to clot at 37°C for 1h, separated by centrifugation at 3600 rpm for 10 minutes, then aliquoted in cryotubes. After swab discharge, homogenization and centrifugation, the viral transport medium was aliquoted. All samples were kept at -80°C at the

National Influenza Reference Laboratory in Bobo-Dioulasso before being transferred to Luxembourg on dry ice for further laboratory analysis. Virological screening and antibody detection RNA extraction from human nasopharyngeal and pig nasal swabs was performed using QIAamp viral RNA minikit (Qiagen, Venlo, the Netherlands). Equine arteritis virus was used as extraction process control for human swabs and detected using the FTD Internal control EAV kit (FastTrack

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Diagnostics, Esch-sur-Alzette, Luxembourg). Measles virus strain MVi/Moscow.RUS/1988 was used as extraction process control for pig swabs and detected using previously published primers and probe (Hubschen et al., 2008). Only samples coming from runs with extraction process control showing the expected Ct value were further analyzed for the presence of influenza A virus by real

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time RT-PCR as previously described (Snoeck et al., 2011).

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The presence of antibodies against influenza A in swine serum was tested by the competitive immuno-enzymatic serology test (ELISA) for antibodies directed against the nucleoprotein

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(IDEXX, Hoofddorp, the Netherlands) as recommended by the manufacturer. Sera reacting in the ELISA were further characterized by virus microneutralization (VN) assays as described before

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(Snoeck et al., 2015). The following representative strains were used in VN assays: pandemic (H1N1) 2009 strain A/Luxembourg/46/2009 (pdm/09); European avian-like swine H1N1 A/swine/Belgium/1/98 (sw/B/98) and A/swine/Gent/112/2007 (sw/G/07); American triple

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reassortant H1N1 A/swine/Iowa/H04YS2/04 (sw/I/04). After heat inactivation at 56°C for 30 min, the sera were screened in triplicate in 2-fold serial dilutions in virus growth medium from 1:20 to 1:2560. Titers were expressed as the reciprocal of the highest serum dilution completely inhibiting virus replication. Samples with no virus neutralization in the 1:20 dilution were given an arbitrary titer of 10. Geometric mean titers (GMT) were calculated from triplicate serum samples.

Statistical analyses Statistical analyses (Chi-square tests and z-test for low proportions; Mann-Whitney rank sum tests) were performed in SigmaPlot version 12.0 (San Jose, CA, USA). The differences were considered significant with p < 0.05. Results No influenza A virus was detected in nasopharyngeal swabs in humans or in pigs. Antibodies

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against influenza A nucleoprotein were found in 6.8% (41/600) of the pig sera (Table 1). Seropositive animals were found in 4/21 (19.0%) semi-intensive farms with within-farm

seroprevalence ranging from 3.3 to 17.4%. Seropositive animals were detected in half of the

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extensive settings (10/20, 50.0%; Table 1). Seroprevalence rates in samples collected in 2016

(8/533, 1.5%) and 2017 (33/67, 49.3%) were significantly different (p<0.001). The median age of

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seropositive animals (24 months) was significantly higher than the median age of seronegative animals (9 months; p<0.001). Mainly pigs of “exotic” breed (pure Large White or, more often,

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crossed breed between imported and local animals such as Korhogo breed) were present in the farms and sampled (549/600, 91.5%). Seroprevalence was significantly higher in pigs of the local

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breed (25/51, 49.0%) compared to those of the exotic breed (16/549, 2.9%; p< 0.001) but the former (median age: 24 months) were also older than exotic pigs (median age: 9 months; p< 0.001). In VN assays, all ELISA positive sera had a GMT ≥ 40 against the pdm/09 strain, with 40/41

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(97.6%) of sera with GMTs ≥ 320 (Figure 1). The prevalence of antibodies against sw/I/04 was lower (36/41; 87.8% with GMTs ≥ 40; p=0.065) compared with pdm/09 and lower titers were observed (0.7-5.3 log2 difference in Ab titers). The prevalence of antibodies against the two European avian-like H1N1 strains was significantly lower for both viruses, compared with pdm/09: 31/41 (75.6% with GMTs ≥ 40; p=0.002) of sera reacted against sw/B/98 and lower titers were observed (0.3-6.3 log2 difference in antibody titers), while 26/41 (63.4% with GMTs ≥ 40;

p<0.001) of sera reacted against sw/G/07 at even lower titers (2-7 log2 difference in antibody titers). Using a more conservative threshold for positivity (GMT ≥ 80), the prevalence of antibodies against pdm/09 was significantly higher than against sw/I/04 (p=0.002), sw/B/98 (p<0.001) and sw/G/07 (p<0.001). Only one serum (the only seropositive specimen from this location by ELISA) originating from a semi-intensive farm had a low antibody titer against pdm/09 strain (GMT = 40) and tested negative against the three other viral strains. In the practice and awareness survey, most farmers (97.3%) declared that they were living on the

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farm, compared to 32.6% of gardeners and none of the veterinarians or slaughterhouse workers (Table 2). Almost all farmers (94.5%) had daily contacts with pigs during feeding or cleaning

activities. Fewer veterinarians (62.6%) and gardeners (38.4%) had daily contacts with live pigs and

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most gardeners (86.7%) were handling feces to be used as fertilizer. All farmers declared that they

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wore no personnel protective equipment except boots (86.3%). Most veterinarians, on the other hand, declared wearing gloves (86.9%), masks (83.8%) and dedicated clothes (85.9%) when

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tending to pigs. Veterinarians (92.3%) followed by slaughterhouse workers (87.0%) were more frequently aware of the possibility of pig-to-human disease transmission, followed by gardeners

Discussion

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(29.1%) and farmers (13.7%; Table 2).

The absence of influenza virus detection in pigs in African countries is not unusual (Couacy-

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Hymann et al., 2012; Snoeck et al., 2015), especially given the short shedding phase and the absence of symptoms upon sampling. Aside from some studies reporting higher (sero)prevalence rates in small cohorts (Adeola et al., 2010; Aiki-Raji et al., 2004; Olaleye et al., 1990), swine exposure to influenza A virus in Africa seems to be limited, especially prior to the spread of pandemic H1N1 (Snoeck et al., 2015). Lower prevalence may potentially be due to lower pig densities compared to other regions (Vincent et al., 2014) combined with suboptimal environmental

factors for the spread of influenza viruses (Couacy-Hymann et al., 2012; Lowen et al., 2007). Sample collection took place during periods of low influenza transmission in humans, as reflected by the absence of detection in human swabs and as also reported by the National Influenza Reference Laboratory (WHO). Although increased influenza transmission tends to occur more frequently in January-March and September-October, influenza seasons in humans vary and are difficult to predict (Sanou et al., 2018). Notwithstanding an acknowledged lack of data, the epidemiology of influenza A virus in swine in

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Africa seems to differ substantially from other continents such as North America, Europe or Asia where enzootic swine influenza strains are ubiquitous. In Africa, avian influenza strains originating from spill-over from concomitant poultry outbreaks were anecdotally transmitted to pigs (El-Sayed

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et al., 2010; Gomaa et al., 2018; Meseko et al., 2018). More importantly, evidence of human

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seasonal H1N1 and H3N2 virus circulation was shown in Nigerian swine (Adeola et al., 2010; Adeola et al., 2016; Aiki-Raji et al., 2004) while pdm/09 has been regularly detected in pig upper

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respiratory tract samples since 2009 (Adeola et al., 2017; Ducatez et al., 2015; Gomaa et al., 2018; Munyua et al., 2018; Njabo et al., 2012; Osoro et al., 2019). Altogether current evidence suggests

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that swine exposure to influenza A in the region is very much influenced by reverse zoonosis events rather than circulation of enzootic African swine influenza strains, a trend also observed in this study.

Pig immunization against influenza A virus is not implemented in Burkina Faso, indicating past

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exposure to a wild type virus in our cohort. Almost all seropositive animals had high antibody titers against pdm/09 and respectively lower titers against the three other swine H1N1 virus strains. These results suggest seroconversion to pdm/09 and cross-reactivity against other virus strains (Garten et al., 2009), similarly to past pig serum cohorts in Nigeria (Meseko et al., 2018; Meseko et al., 2016; Snoeck et al., 2015), Cameroon (Njabo et al., 2012; Snoeck et al., 2015), Egypt (Gomaa et al., 2018) and Kenya (Munyua et al., 2018).

All pdm/09 strains sequenced previously in pigs in Africa were shown to be closely related to recent strains circulating in the human population, which suggested recent human-to-swine virus transmission (Adeola et al., 2017; Ducatez et al., 2015; Gomaa et al., 2018; Munyua et al., 2018), and a similar event could also have happened here. In Burkina Faso, pdm/09 was only detected in two instances in 2016 in the framework of the national sentinel surveillance, while seasonal H3N2 dominated. In contrast, pdm/09 was detected from February till May 2017 and seasonal H3N2 was not reported (WHO). These seasonal differences in the human population might also partially

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explain the difference in seroprevalence between swine samples collected in 2016 and 2017 in our study. Nevertheless, the hypothesis of sustained swine-to-swine transmissions of pdm/09 cannot be ruled out. Indeed, the existence of a lineage of pdm/09 maintained in swine was shown in France

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for instance (Chastagner et al., 2018). To characterize the relative importance of each phenomenon, future studies would benefit from sampling during various periods of the year, including during

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acute seasonal influenza virus transmission periods. The distinction between sustained or recurrent

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transmissions and their frequency will have differential implications for preventive measures against the introduction and spread of influenza viruses in pig populations.

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One serum had low antibody titers (GMT≤40) against the four strains tested. Occasional exposure to other viral strains Beside pdm/09 can therefore not be excluded. Seroconversion to human seasonal H3N2 was suspected in small pig cohorts in neighboring countries (Adeola et al., 2010; Adeola et al., 2016). Nevertheless, a regional or continental effort towards virological screening,

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complete virus genome characterization as well as country-wide serological studies in pigs are warranted in order to properly understand the epidemiology of influenza in swine in Africa. Since influenza A viruses in swine and human hosts are connected, understanding practices that may influence virus transmission is important to establish prevention measures. The practices and knowledge survey analysis highlighted a lack of awareness regarding risk of occupational pathogen exposure in certain groups, especially farmers. It pointed out that inter-host influenza virus

transmission would more likely arise from contacts with farmers who wear no protective equipment and have increased frequency of contact. These observations concur with a lower seroprevalence in (semi-)intensive compared to extensive rearing systems where increased human proximity is expected. In contrast, veterinarians are better aware and protected and are seldom called in low income settings. Slaughterhouse workers likely have a lower exposure risk (Myers et al., 2006), and little to no impact on human-to-swine virus transmission, yet their degree of awareness was rather high. Both animal and human health, as well as income, would benefit from improved awareness of

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people with exposure to live pigs regarding (reverse) zoonotic transmission of influenza A virus. In conclusion, swine exposure to influenza A viruses, including pdm/09, is still under-reported in Africa while biosecurity measures in pig husbandry remain suboptimal. Awareness campaigns

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targeting swine workers, and farmers in particular, should be initiated to promote simple

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recommendations such as seasonal vaccination, wearing respiratory masks or refraining from contact during influenza-like illness episodes. A regional effort towards virological screening,

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complete virus genome characterization as well as large-scale serological studies in pigs are warranted in order to properly understand the epidemiology of influenza in swine in Africa.

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Conflict of interest statement

The authors declare no conflict of interest. Acknowledgements

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The authors wish to thank J.-F. Bonkoungou, the agents of the regional breeding laboratory and the authorities in charge of animal resources in the Hauts-Bassins region for their collaboration. The authors deeply acknowledge C.P. Muller for his contribution in funding acquisition. We thank K. Van Reeth from Faculty of Veterinary Medicine, Ghent University, Belgium, for providing swine influenza strains and control sera.

This study was supported by the Luxembourg Ministry of Foreign and European Affairs, the Luxembourg Ministry of Higher Education and Research and Luxembourg Institute of Health [grants MAE-IV and MAE-V]. D. Tialla was also supported by a PhD fellowship and trainings

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funded by the same sources. The funders had no involvement in the study design or its publication.

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WHO. FluNet.https://www.who.int/influenza/gisrs_laboratory/flunet/en/

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Figure legend

Fig. 1. Geometric mean titer distribution of serum neutralizing antibodies against four H1N1

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influenza A viruses

Table 1. Description of sampled pig herds and prevalence of antibodies against the nucleoprotein of influenza A virus (as detected by ELISA) Average

Settings

settings sampled

Extensive

23.3 [11-36]

21

250.8 [44-504]

No. of

sample

size/farm

pigs

size/herd

[range]

sampled [range]

47.2 [8-518]

40

n.a.

settingsa sera (%)

42

2.0 [1-3]

17 (42.5)

10 (50.0) 4 (19.0)

7 (1.3)

50]

30 600

n.a.

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Seropositive setting was defined as a setting hosting at least one seropositive animal.

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(%)

25.2 [4530

n.a.

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seropositive positive

1002]

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poultry flock

234.5 [12-

Intensive

No. of No. of Ab

[range]

20

Slaughterhouse 1

Average

herd size/farm

(Semi-)

Total

Total

Average pig

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No. of

17 (56.7) 41 (6.8)

1 (100.0)

Table 3. Overview of practices and awareness survey Variables

No. of people (%) Farmers

Veterinaria Gardeners

Slaughterho

(n=73)

ns

use workers

(n=86)

Living on a farm

(n=100)

71 (97.3)

0 (0)

Daily

69 (94.5)

62 (62.6)

Weekly

4 (5.5)

25 (25.3)

Monthly or less

0 (0.0)

12 (12.1)

ro of

(n=99) 28 (32.6)

None

0 (0.0)

0 (0.0)

Frequency of contact with pigs

100 (100)

28 (32.6)

0 (0)

10 (11.6)

0 (0)

15 (17.4)

0 (0)

0 (0.0)

0 (0.0)

0 (0)

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33 (38.4)

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Type of activities leading to

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contact with pigs/pig derived products

72 (98.6)

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Feeding

0 (0)

Barn cleaning/feces handling

72 (98.6)

0 (0.0)

72 (83.7)

0 (0)

Veterinary care

0 (0.0)

98 (99.0)

0 (0.0)

0 (0)

63 (86.3)

85 (85.9)

35 (40.7)

0 (0)

Dedicated clothes

0 (0.0)

85 (85.9)

1 (1.2)

0 (0)

Gloves

0 (0.0)

85 (85.9)

1 (1.2)

0 (0)

Mask

0 (0.0)

83 (83.8)

1 (1.2)

0 (0)

10 (13.7)

92 (92.9)

25 (29.1)

87 (87.0)

Personnel protective equipment

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Boots

Zoonosis awareness

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