ABSTRACT
For an analysis of the prevalence of influenza A viruses (IAVs) circulating in chickens and their farmers in the Ashanti region, Ghana, we examined 2,400 trachea and cloaca swabs (chickens) and 102 oropharyngeal swabs (farmers) by qRT-PCR. Sera from 1,200 (chickens) and 102 (farmers) were analysed for IAV antibodies by ELISA and haemagglutination inhibition (HI). Avian influenza virus (AIV) was detected in 0.2% (n = 5) of chickens but not farmers. Virus detection was more pronounced in the cloacal (n = 4, 0.3%) than in tracheal swabs (n = 1, 0.1%). AIV antibodies were not detected in chickens. Two farmers (2.0%) tested positive to human seasonal IAV H1N1pdm09. Sixteen (15.7%) farmers tested seropositive to IAV of which 68.8% (n = 11) were due to H1N1pdm09-specific antibodies. AIV H5- or H7-specific antibodies were not detected in the farmers. Questionnaire evaluation indicated the rare usage of basic personal protective equipment by farmers when handling poultry. In light of previous outbreaks of zoonotic AIV in poultry in Ghana the open human-animal interface raises concern from a OneHealth perspective and calls for continued targeted surveillance.
Introduction
Worldwide, influenza A viruses (IAVs) are important veterinary and public health pathogens causing substantial morbidity and mortality in varying species including humans and poultry [Citation1,Citation2]. The viruses are facing host restriction barriers, but interspecies transmission with variable sequelae can occur: (i) abortive infection, (ii) productive infection associated or not with clinical disease, (iii) adaptation to new host species with secondary virus transmission. For avian influenza viruses (AIVs) high pathogenic (HP) and low pathogenic (LP) phenotypes have been described and both can harbour zoonotic propensity. The impact of HPAIVs on livelihood and food security especially of low-income countries can be immense due to the highly lethal course of disease especially in gallinaceous poultry [Citation1]. Outbreaks of LPAIVs in gallinaceous poultry do not necessarily receive control responses in contrast to HPAIV outbreaks. However, when allowed to continuously circulate in gallinaceous poultry, LPAIV of subtypes H5 and H7 can mutate to notifiable HPAIV; other subtypes may reassort with other IAVs of avian, porcine or human origin to generate strains with extended zoonotic and even human pandemic potential [Citation3–Citation5]. Sporadic human infections with AIVs have been reported worldwide with higher incidences among individuals in direct contact with infected poultry, contaminated poultry products and/or poultry environment [Citation6–Citation9]. There has been a growing interest in AIV infections in Africa following the introduction of HPAIV H5N1 in gallinaceous poultry in 2006 [Citation1], contributing to the identification of different AIV subtypes with known and unknown zoonotic propensities in birds on the continent [Citation5,Citation10–Citation14]. Simultaneously, evidence of AIV infections, exposures and death among humans in regular contact with poultry on the continent have also increased [Citation9,Citation15,Citation16].
In Ghana, outbreaks of zoonotic HPAIV H5N1 (clade 2.2 and 2.3.2.1c) in poultry have been reported with no human deaths [Citation9,Citation17]. Studies focusing on active infection after the first outbreak (in 2007), recognised an increased risk of zoonotic transmission due to poor implementation of biosecurity and biosafety practices among poultry handlers [Citation18–Citation20].
The Ashanti region is the second-largest commercial poultry-producing region in Ghana. An HPAIV H5N1 outbreak was recorded in the region only during the second HPAI outbreak in the country in 2015. The region is a hub for trading live poultry and/or poultry products to other parts of the country and to neighbouring countries. Little is known about AIV infections in commercial poultry and much less of poultry handlers within the area. AIV was not detected in surveillance carried out in commercial poultry before the first H5N1 outbreak in the country, and in backyard poultry in military barracks in the region after the first outbreak [Citation20]. We performed a cross-sectional study to determine the prevalence of IA viruses in commercial chickens and their farmers within the Ashanti region of Ghana. This will contribute to our understanding of influenza at the human-animal interface in the region and aid to develop IAV control strategies to prevent infections in poultry and humans.
Materials and methods
Ethics and sampling
Ethical approval was obtained from the Council for Scientific and Industrial Research (RPN 001/CSIR-IACUC/2016), Ghana, and Ärztekammer Hamburg (PV5296), Germany. Between April 2016 to February 2017 tracheal and cloacal swabs and blood samples (2 mL) were collected from 1,200 clinically healthy chickens raised exclusively in-house on 76 commercial chicken farms in the Ashanti region. An oropharyngeal swab and a blood sample (2 mL) were obtained from 102 farmers from 39 of these farms. None of the farmers had symptoms suggestive of any respiratory illness at the time of sampling. Swabs were collected into viral transport medium [Citation21] and transported on ice to the laboratory. Questionnaires were used to collect relevant farm and farmer data.
Laboratory analysis
RNA was isolated from swabs (QIAamp viral RNA mini kit, Germany) and tested for influenza A Matrix-specific gene by qRT-PCR [Citation22]. All positive samples were subjected to direct subtyping of all AIV subtypes [Citation23,Citation24]. Additionally, human samples were tested for seasonal influenza viruses of subtypes H1 and H3 by qRT-PCR [Citation24]. Viral isolation in embryonated chicken eggs and MDCK cells of positive samples was attempted. ELISA was used to test sera for IAV antibodies (IDEXX AI MultiS-Screen kit, chicken; Serion IgG ELISA kit, human). Hemagglutination inhibition (HI) assay was used to test ELISA positive sera for avian H5 and H7 and seasonal H1 antibodies (A/tky/England/647/1977 (H7N7); A/Teal/England 7394-2805/2006 (H5N3); source: European Reference Laboratory for Avian Influenza, Weybridge, UK, and H1N1pdm in-house control strain of FLI, A/Germany/R26/2010 (H1N1pdm)). Frequency and percentages were computed for categorical variables. Median and interquartile range (IQR) were computed for continuous variables. The point prevalence along with the 95% confidence interval (CI) was estimated. Data were analysed with STATA 14.
Results
Influenza a prevalence on poultry holdings
Based on questionnaire analyses, most farms (n = 55, 72.4%) had up to 5,000 chickens and the majority (n = 72, 94.7%) kept only layers. Majority of farms (n = 69, 90.8%) reported at least one episode of respiratory infection among the chickens between 3 weeks to 4 months prior to sampling. Nearly all farms (n = 75, 98.7%) retailed their spent layers live, and table eggs at the farm gate. Vaccination against AIV is not practiced in Ghana. AIV was detected in 0.2% (n = 5/2400, 95% CI = 0.19–0.23) of chicken swabs. Viral RNA was detected on 5.3% (n = 4/76) of farms. Four out of five of AIV positive samples were of cloacal origin (). All positives were detected in layers. The quantitation cycle (Cq) value of all positives ranged from 35 to 38 indicating a very low virus load. The direct subtyping attempt was unsuccessful. Viral isolation attempts failed. AIV antibodies were not detected in any of the 1,200 chicken sera ().
Influenza a prevalence among chicken farmers
The median age of farmers was 25 years (IQR = 22.0–35.0) and most (n = 74, 72.5%) had worked at the present farm for more than 1 year. Only 2 (2.0%) reported to wear a surgical face mask and none reported to wear gloves when working.
IAV RNA was detected in two swabs from humans. Both were subtyped as H1N1pdm09. Sixteen farmers had IAV antibodies. AIV H5- and H7-specific antibodies were not detected. Antibodies to H1N1pdm09 were detected in 10.8% (11/102) of total sera analyzed (), and formed 68.8% (11/16) of seropositive samples. All AIV positive farms had a farmer who tested positive to either H1N1pdm09 virus or antibody.
Discussion
The study could not find evidence for endemic circulation of AIV in apparently healthy commercial chickens raised exclusively in-house on farms in the Ashanti region shortly before and during the study period. This is highlighted by the lack of AIV antibodies in any of the chickens examined; following an AIV infection antibodies in layer chickens are expected to be detectable at least 4–6 months after recovery. Thus, past episodes of respiratory disease in layers, as reported by farmers, are most likely unrelated to AIV infections. However, very few cases of active shedding of clinically healthy chickens mostly through faeces were detected. This suggests rare sporadic infection with LPAIV. Subtyping of these viruses was precluded by the very low virus load present in the samples. Previous reports from Ghana likewise did not detect active AIV infection in healthy poultry [Citation18–Citation20] and a low prevalence was reported from Kenya [Citation25].
In several African countries, in contrast, LPAIV alone or in co-infections with other avian pathogens have caused high morbidity, drop in egg production and mortality [Citation26–Citation28]. Interestingly, LPAIV H9N2 in co-infection with infectious bronchitis virus (IBV) caused a significant drop in egg production and high mortality on several layer farms in the Ashanti region a few months after the current study had been finalised. The current study suggests that this virus has not previously circulated in the farms visited but likely was recently introduced into the chicken population, highlighting the consequences of low biosafety measures on farms [Citation29]. Unrestricted moving of AIV-infected live chickens between farms and markets may have played a key role in spreading LPAIV in the country and increase public health risks [Citation30]. The origin of the H9N2 virus later on detected remained unclear but the close phylogenetic relationship to viruses circulating endemically in poultry in several North African countries suggested transboundary i ncursions related to poultry trade [Citation29]. Therefore, raising biosafety standards on poultry farms would be a basic precondition to limit economic losses due to infectious diseases. Controlling trade-related transports of live poultry may further aid in reducing the risk of viral spread. This would be particularly important in case zoonotic AIVs are encountered. Interestingly, the H9N2 viruses causing the reported incursion into Ghana are members of the zoonotic G1 lineage that previously caused human infections in Egypt [Citation31].
Members of the Asian HPAIV H5 lineage with mammalian receptor affinity caused sporadic outbreaks in chicken farms in the Ashanti region, in 2015, 2016 and 2018 [Citation32,Citation33]. The rapid response of the veterinary services of Ghana significantly reduced viral spread and possible contact of farmers with the virus. The absence of H5- and H7-specific antibodies in the farmers despite frequent and long contact to poultry rules out infection with these zoonotic pathogens [Citation7]. In contrast, infections, acute and past, with seasonal human IAV subtype H1N1 was detected. In Nigeria, Cameroon, and Egypt where H5 and H7 antibodies have been detected in poultry workers, the corresponding avian viruses were observed to have circulated for longer periods and affected more poultry holdings, increasing the net exposure risk of poultry workers with possibly infected poultry [Citation15,Citation16,Citation34]. Nevertheless, farmers’ compliance with certain basic biosafety practices were largely poor as noted previously in other parts of the country [Citation18–Citation20] and therefore the risk of exposure to zoonotic AIVs such as LPAIV H9N2 [Citation29] and other non-viral avian pathogens remains high. Co-circulation of IAVs in farmers and their chickens increases the risk of generating reassortants. Regular surveillance of IAVs at the human-animal interface in poultry production for early detection and effective control of these emerging zoonotic and potentially pandemic IAVs would be highly desirable.
Acknowledgments
We are grateful to the farmers who participated in the study. Thanks are also due to Sina Ramcke, Diana Parlow and Aline Maksimov at FLI for technical support. The Open Access Fund of the Leibniz Association funded the publication of this article
Disclosure statement
No potential conflict of interest was reported by the authors.
Additional information
Funding
Notes on contributors
Ayim-Akonor Matilda
Ayim-Akonor Matilda is an early career scientist with the Council for Scientific and Industrial Research-Animal Research Institute, Ghana and a PhD student at the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. Her research focuses on the epidemiology of infectious diseases of livestock and poultry as well as zoonotic diseases both of national and international interest.
May Juergen
Juergen May is Full Professor for Epidemiology of Tropical Diseases at the University of Hamburg, Member of the Board of Directors at the Bernhard Nocht Institute for Tropical Medicine (BNITM), and Head of the Department Infectious Disease Epidemiology. His research focus is the epidemiology and control of infectious diseases in sub-Saharan Africa.
Ralf Krumkamp
Ralf Krumkamp is senior epidemiologist at the Bernhard Nocht Institute for Tropical Medicine, Germany. His main research interests are transmission dynamics and the control of infectious diseases.
Harder Timm
Timm C. Harder graduated as a veterinarian and is now head of the German National Reference Laboratory for Avian Influenza at the Friedrich-Loeffler-Institute (FLI, Federal Institute for Animal Health), Isle of Riems, Germany. The laboratory is an active member of international networks of the World Health Organization for Animal Health (O.I.E.) and the Fodd and Agriculture Organization (FAO) of the UN for research and diagnosis on animal influenza.
Mertens Eva
Eva Mertens is an epidemiologist in the infectious disease epidemiology department at the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany. Her background is in virology and her interests are in zoonoses and infectious disease control.
References
- Fasanmi OG, Odetokun IA, Balogun FA, et al. Public health concerns of highly pathogenic avian influenza H5N1 endemicity in Africa. 2017. Veterinary World, 10(10):1194–1204.
- Iuliano AD, Roguski KM, Chang HH, et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet. [Internet]. 2018 Mar 31 [cited 2018 May 4];391(10127):1285–5. Available from http://www.ncbi.nlm.nih.gov/pubmed/29248255
- Castrucci MR, Donatelli I, Sidoli L, et al. Genetic reassortment between avian and human influenza a viruses in. Pigs I Virology. [Internet]. 1993 Mar 1 [cited 2019 Nov 5];193(1):503–506. Available from https://www.sciencedirect.com/science/article/abs/pii/S0042682283711554
- Hagag NM, Erfan AM, El-Husseiny M, et al. Isolation of a novel reassortant highly pathogenic avian influenza (H5N2) Virus in Egypt. Viruses. [Internet]. 2019 Jun 18 cited 2019 Aug 30;11(6):565. Available from http://www.ncbi.nlm.nih.gov/pubmed/31216712
- Naguib MM, Verhagen JH, Samy A, et al. Avian influenza viruses at the wild-domestic bird interface in Egypt. Infect Ecol Epidemiol. [Internet]. 2019 [cited 2019 Aug 3];9(1):1575687. Available from http://www.ncbi.nlm.nih.gov/pubmed/30815236
- Puzelli S, Rizzo C, Fabiani C, et al. Influenza A(H7N7) virus among poultry workers, Italy, 2013. Emerg Infect Dis. [Internet]. 2016 [cited 2019 Aug 27];22(8):1512–1513. Available from http://www.ncbi.nlm.nih.gov/pubmed/27434025
- Gao R, Cao B, Hu Y, et al. Human infection with a novel avian-origin influenza A (H7N9) Virus. N Engl J Med. [Internet]. 2013 May 16 [cited 2018 May 4];368(20):1888–1897. Available from http://www.nejm.org/doi/10.1056/NEJMoa1304459
- Zhou L, Liao Q, Dong L, et al. Risk factors for human illness with avian influenza A (H5N1) virus infection in China. J Infect Dis. [Internet]. 2009 Jun 15 [cited 2019 Aug 21];199(12):1726–1734. Available from http://www.ncbi.nlm.nih.gov/pubmed/19416076
- World Health Organization. Cases deaths [Internet]. 2019. 2019 [cited 2019 Aug 28]. p. 3. Available from: https://www.who.int/influenza/human_animal_interface/2019_06_24_tableH5N1.pdf?ua=1
- Coker T, Meseko C, Odaibo G, et al. Circulation of the low pathogenic avian influenza subtype H5N2 virus in ducks at a live bird market in Ibadan, Nigeria. Infect Dis Poverty. [Internet]. 2014 [cited 2019 Aug 7];3(1):38. Available from http://www.ncbi.nlm.nih.gov/pubmed/25671118
- Zecchin B, Minoungou G, Fusaro A, et al. Influenza A(H9N2) Virus, Burkina Faso. Emerg Infect Dis. [Internet]. 2017 Dec [cited 2019 Aug 7];23(12):2118–2119. Available from http://www.ncbi.nlm.nih.gov/pubmed/28980894
- FULLER TL, DUCATEZ MF, NJABO KY, et al. Avian influenza surveillance in Central and West Africa, 2010–2014. Epidemiol Infect. [Internet]. 2015 Jul 22 [cited 2019 Aug 27];143(10):2205–2212. Available from http://www.ncbi.nlm.nih.gov/pubmed/25530320
- Abolnik C, Olivier A, Reynolds C, et al. Susceptibility and status of avian influenza in ostriches. Avian Dis. [Internet]. 2016 Feb 10 [cited 2019 Aug 7];60(1s):286. Available from http://www.ncbi.nlm.nih.gov/pubmed/27309069
- Couacy-hymann E, Kouakou VA, Aplogan GL, et al. Surveillance for infl uenza viruses in poultry and swine, West Africa. Emerg Infect Dis. 2012;18(9):1446–1452.
- Gomaa MR, Kandeil A, Kayed AS, et al. Serological evidence of human infection with avian influenza A H7virus in Egyptian poultry growers. PLoS One. Internet]. 2016;11(6):1–10. Available from http://dx.doi.org/10.1371/journal.pone.0155294
- Monamele CG, Karlsson EA, Vernet M-A, et al. Evidence of exposure and human seroconversion during an outbreak of avian influenza A(H5N1) among poultry in Cameroon. Emerg Microbes Infect. [Internet]. 2019 [cited 2019 Aug 28];8(1):186–196. Available from http://www.ncbi.nlm.nih.gov/pubmed/30866772
- Tassoni L, Fusaro A, Milani A, et al. Genetically different highly pathogenic avian influenza A(H5N1) viruses in West Africa, 2015. Emerg Infect Dis. [Internet]. 2016 [cited 2019 Sep 30];22(12):2132–2136. Available from http://www.ncbi.nlm.nih.gov/pubmed/27389972
- Agbenohevi PG, Odoom JK, Bel-Nono S, et al. Biosecurity measures to reduce influenza infections in military barracks in Ghana. BMC Res Notes. 2015;8(1):1–8.
- Burimuah V, Ampofo WK, Awumbila B, et al. The evaluation of domestic ducks as potential reservoir of avian influenza virus in post Hpai H5n1 outbreak area, sunyani municipality, brong ahafo region Of Ghana. African J Infect Dis. [Internet]. 2016 cited 2019 Aug 28;10(2):134–145. Available from http://www.ncbi.nlm.nih.gov/pubmed/28480449
- Odoom JK, Bel-Nono S, Rodgers D, et al. Troop education and avian influenza surveillance in military barracks in Ghana, 2011. BMC Public Health. [Internet]. 2012;12:957.Available from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3534292&tool=pmcentrez&rendertype=abstract
- Eisfeld AJ, Neumann G, Kawaoka Y. Influenza A virus isolation, culture and identification. Nat Protoc. [Internet]. 2014;9(11):2663–2681.
- Hoffmann B, Depner K, Schirrmeier H, et al. A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses. J Virol Methods. [Internet]. 2006 Sep [cited 2018 Oct 29];136(1–2):200–209. Available from http://www.ncbi.nlm.nih.gov/pubmed/16806503
- Hoffmann B, Hoffmann D, Henritzi D, et al. Riems influenza a typing array (RITA): an RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses. Sci Rep. [Internet]. 2016;6(February):27211. Available from http://www.ncbi.nlm.nih.gov/pubmed/27256976%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4891686
- Henritzi D, Zhao N, Starick E, et al. Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs. Influenza Other Respi Viruses. [Internet]. 2016 [cited 2018 Jul 5];10(6):504–517. Available from http://www.ncbi.nlm.nih.gov/pubmed/27397600
- Munyua P, Onyango C, Mwasi L, et al. Identification and characterization of influenza A viruses in selected domestic animals in Kenya, 2010-2012. PLoS One. [Internet]. 2018 [cited 2019 Oct 1];13(2):e0192721. Available from http://www.ncbi.nlm.nih.gov/pubmed/29425232
- Sid H, Benachour K, Rautenschlein S. Co-infection with multiple respiratory pathogens contributes to increased mortality rates in algerian poultry flocks. Avian Dis. [Internet]. 2015 Sep [cited 2019 Sep 30];59(3):440–446. Available from http://www.ncbi.nlm.nih.gov/pubmed/26478165
- Kammon A, Heidari A, Dayhum A, et al. Characterization of avian influenza and newcastle disease viruses from poultry in Libya. Avian Dis. [Internet]. 2015 Sep [cited 2019 Sep 30];59(3):422–430. Available from http://www.ncbi.nlm.nih.gov/pubmed/26478162
- El Houadfi M, Fellahi S, Nassik S, et al. First outbreaks and phylogenetic analyses of avian influenza H9N2 viruses isolated from poultry flocks in Morocco. Virol J. [Internet]. 2016 [cited 2019 Sep 30];13(1):140. Available from http://www.ncbi.nlm.nih.gov/pubmed/27527708
- Awuni JA, Bianco A, Dogbey OJ, et al. Avian influenza H9N2 subtype in Ghana: virus characterization and evidence of co-infection. Avian Pathol. 2019;1–7. DOI:10.1080/03079457.2019.1624687
- Mensah-bonsu A, Rich KM Ghana ’ s Poultry Sector Value Chains and the Impacts of HPAI.2010;(Oct)
- Gomaa MR, Kayed AS, Elabd MA, et al. Avian influenza A(H5N1) and A(H9N2) seroprevalence and risk factors for infection among Egyptians: a prospective, controlled seroepidemiological study. J Infect Dis. [Internet]. 2015 May 1 [cited 2019 Sep 30];211(9):1399–1407. Available from http://www.ncbi.nlm.nih.gov/pubmed/25355942
- Asante IA, Bertram S, Awuni J, et al. Highly pathogenic avian influenza A(H5N1) virus among poultry, Ghana, 2015. Emerg Infect Dis. 2016;22(12):2209–2211.
- Services-Ghana DKMKG (Veterinary. Follow-up report No. 34 (Final report) Outbreak details [Internet]. Vol. 34, Oie, UPDATE ON HIGHLY PATHOGENIC AVIAN INFLUENZA IN ANIMALS. 2017 [cited 2019 Sep 30]. Available from: https://www.oie.int/wahis_2/temp/reports/en_fup_0000021647_20161124_170217.pdf
- Okoye J, Eze D, Krueger WS, et al. Serologic evidence of avian influenza virus infections among Nigerian agricultural workers. J Med Virol. [Internet. 2013 Apr [cited 2019 Aug 28];85(4):670–676. Available from http://www.ncbi.nlm.nih.gov/pubmed/23400898