We compiled an updated global catalogue of parasites in cephalopods. Data were used to assess changes in taxonomic distinctness of parasites over two centuries and across the world’s oceans, to quantify turnover and nestedness components of parasite β-diversity, and to attempt estimating their γ-diversity at a global scale. A total of 309 parasites infecting 164 cephalopods were found. We hypothesize that this diversity counts for less than half the potential parasite richness in this molluscan taxon. Taxonomic breadth of parasites was significantly above expectations from null models for Mediterranean Sea and NE Atlantic Ocean, whereas the opposite occurred for NW Pacific Ocean, where a few closely related genera characterized the parasite pool. β-diversity of parasites was very high and dominated by turnover, except for the Atlantic Ocean where a nested pattern among sub-basins emerged. Taxonomic relatedness of parasite species remained substantially unchanged through time, but species replacements largely occurred over the last two centuries. Our findings highlighted potential hotspots of taxonomic distinctness in cephalopod parasites, geographic regions deserving future research, and the need for a deeper understanding of the magnitude of marine parasite diversity, their biogeography, and their role in marine ecosystems. Our global overview may represent a baseline step for future advances in this direction. Cephalopods also play a crucial role in marine food webs7, both as active predators of a wide range of marine species and as prey for fish, birds and mammals.With more than 800 recognized species, cephalopods inhabit a variety of marine habitats worldwide, from the intertidal zone to the deep ocean. They include species that greatly differ in terms of habitat preference, sociability  and biological traits . Several of these species are of interest to fisheries and aquacultureOctopus spp., Opalinopsidae ciliates, with members of the genus Chromidina parasitic in the renal appendages, and Opalinopsis parasitic in the liver and intestine of several Octopoda, Sepiida and Oegopsida worldwide. Coccidians of the genus Aggregata are also frequent, occurring with very high prevalence in the digestive tract of different species of Decapodiformes and Octopodiformes9. Members of the phylum Dicyemida, traditionally placed among the mesozoans due to their simple body plan, and recently placed among Spiralia10, are common but exclusively found in the renal sac of benthic cephalopods where they usually occur in heavy infections. Among metazoans, many taxa of arthropod and helminth parasites are shared between cephalopod and fish hosts, displaying low host specificity11, although there are examples of high specificity, such as copepods of the subfamily Cholidyinae, which includes free-living species found to infect exclusively cephalopods12.Cephalopods harbour a variety of protozoan and metazoan parasites, which are normally acquired from the environment and through the food web. Common protozoan parasites include Ancistrocomidae ciliates, parasitizing the skin and gills of 16. In cephalopods, evidence suggested that the ecological niche of species is the main factor shaping its parasitic fauna17, although geographic factors may also play a crucial role in differentiating parasite species composition and abundance18. Climate change and human impacts may affect the abundance and distribution of marine parasites directly and indirectly, by influencing their free-living stages, migration patterns of their hosts19 or their abundance20. This is particularly true for heteroxenous, trophically transmitted parasites, such as anisakids, whose successful diffusion requires a wide availability of hosts throughout their life cycle and the stability of marine trophic webs21.Parasite diversity is often the result of multiple factors, such as habitat features, environmental changes, food preferences, migration of hosts, age and maximum size reached by the host, gregariousness or solitary behaviour22. The increased use of molecular biology allowed resolving some uncertainties in taxonomic identifications related to the small size of parasitic organisms, and to unveil the presence of cryptic species and morphologically different life stages25. However, information on parasite diversity in cephalopods is still far from being up to date and summarized26.During the last decades, research in parasitology benefited from the development of new technologies and investigation techniques, which in part counterbalanced the decline in the number of expert taxonomists27. Specifically, we aimed (1) to provide a picture of the magnitude, distribution, and historical trends of parasite diversity in cephalopods at a global scale. Also, we used taxonomic distinctness indices and multivariate analysis (2) to assess potential changes in taxonomic relatedness and species composition of parasites in the last two centuries, (3) to test whether the taxonomic relatedness of parasite species varied across the world’s oceans, (4) to quantify β-diversity of parasite assemblages among different biogeographic areas and periods, and (5) to explore putative host-specific associations for most widespread cephalopod genera of high ecological and commercial value. Finally, we exploited the full record to (6) attempt an estimate of γ-diversity of parasites in cephalopods worldwide.In this study, we attempted for the first time to provide a comprehensive synthesis of parasites diversity in cephalopod hosts by compiling a list of host-parasite associations since the first documented parasitic infection in a cephalopod host, dating back to the seventeenth centuryA total of 492 articles were selected, reporting 695 host-parasite associations. Records span from 1684  to 2017. The database includes associations involving cephalopods of the subclass Coleoidea  and only 1 association with one species in the subclass Nautiloidea. Parasites belonged to 11 phyla, although six phyla encompassed 98% of host-parasite associations , followed by Nybelinia lingualis Dollfus, 1929 (11), and Tentacularia coryphaenae Bosc, 1802 (11), and nematodes Anisakis simplex  (22) and Anisakis physeteris  (9). A higher number of parasite species was reported infecting benthic-demersal cephalopods such as Octopoda, Sepiida and Myopsida squids (263 parasite taxa) as compared to pelagic Oegopsida squids (88 parasite taxa).The most represented parasitic phyla in terms of number of records were Platyhelminthes (275), Dicyemida (174) and Nematoda (101). Parasites infecting the highest number of cephalopod species were cestodes, with the larval form The spatial distribution of records covers, although unevenly, almost all seas and oceans Fig. a. The hi11, 203 new host-parasite associations were found, among which 62 included the description of new parasite species: several apicomplexans of the genus Aggregata, copepods , and many dicyemids. On the other hand, a considerable amount of parasite species (150) disappeared from records after 1990. These species were mostly dicyemids (40%), cestodes (16%), trematodes (12%) and copepods (11%). Missing parasites also include species, such as for instance the nematodes Pseudoterranova decipiens  Gibson, 1983 and Hysterothylacium reliquens  Deardorff and Overstreet, 1981, which disappeared from records but were found in other host organisms .The number of host-parasite records showed a large increase during the first half of the twentieth century and until the end of 1980s and declined in the last 3 decades, though the number of parasite species recorded remained almost unchanged Fig. b. When cΔ+ and Λ+ values, except for MED, NEA, and NWP .The pool of parasite species characterizing different regions did not show a significant departure from random expectations of NWP Fig. . The forNWP Fig. . Tests oΔ+ and Λ+ did not detect significant changes in taxonomic structure of parasites during the last two centuries , indicating that the parasite species in each period were random subsets of the total pool of parasites in cephalopods.Randomization tests on ies Fig. . TaxonomP < 0.01 in all cases), suggesting that biogeographic peculiarities in parasites species could be driven by differences in host identities among regions and species-specificity of parasite-host associations.β-diversity among parasite assemblages from different regions was very high, with an overall dissimilarity of 97%. The contribution of species turnover to β-diversity was dominant (94%), indicating that cephalopods from different regions were infected by completely different pools of parasites, whereas the contribution of nestedness was negligible (3%). The parasite pool from the Southeast Atlantic Ocean (SEA) was an exception to this pattern, since its dissimilarity with NEA and SWA was mostly due to nestedness , indicating that parasite species from SEA were in large part a nested subset of those found in the other sub-basins of the Atlantic Ocean  and aligned with the corresponding dissimilarity among host assemblages (86%). However, while the contribution of turnover prevailed in determining parasite dissimilarity through time , turnover and nestedness equally participated to the overall temporal change in hosts , indicating that the replacement of parasite species through time could not be merely related to changes in species composition of hosts , and specifically to the two genera Dicyema and Dicyemennea, whereas the remaining 40% of species was distributed among 6 phyla .When analysing the taxonomic structure of parasite assemblages in the five target cephalopod genera, we found that the pool of parasites hosted by the genera ns Table . In contns Table , indicatOctopus also hosted the highest number of parasite species, followed by the genus Sepia, whereas genera Todarodes and Loligo had lower parasite richness. Species composition of parasite assemblages greatly differed among the four investigated genera, with an overall Jaccard dissimilarity of about 95%, which was mostly due to turnover (91%) with a minor contribution of nestedness (> 4%). Only four parasite species were common to the five genera and, namely, A. simplex, Phyllobothrium sp., Chromidina elegans , and N. lingualis.The genus Aggregata sp., Anisakis sp., Contracaecum sp., Pennella sp., Chromidina sp., Derogenes varicus  Looss, 1901, Hysterothylacium sp., Nybelinia sp., and S. pleuronectis were shared among three genera, whereas the remaining parasite species (194) were associated to two genera or were specific of a single genus.The extrapolation of global parasite richness over 800 species of extant cephalopods, based on parasite associated to the 164 cephalopods species recorded, led to estimate a potential number of parasite species equal to 710 (± 121).33. Our findings highlighted that the high diversity of their hosts in these regions reflected into the diversification of cephalopod parasites. The high taxonomic distinctness of parasites in NEA and Med, which is not related to an analogous geographic pattern in their hosts, reinforce the evidence that these two basins can be considered at least as hotspots of parasite phylogenetic diversity. NWP, in contrast, had a significantly low value of taxonomic distinctness due to the high recurrence of species belonging to Dicyemidae. This disproportionate contribution of dicyemid to parasites diversity could depend on the activity of local research groups specialized in this taxon . However, the high number of dicyemid species recorded notwithstanding the relatively limited number of host species analysed, seems to indicate that this group of parasites may be more common than previously thought in cephalopods, remaining often undescribed due to the lack of specific taxonomic expertise.Gaps in geographic representativeness of records make any generalization on spatial distribution of parasite diversity still premature. However, our analyses highlighted some emergent patterns, over and beyond potential bias due to the uneven occurrence of studies among regions, which could help setting next research priorities in the field. The geographical distribution of records showed areas of intense research on cephalopods parasites, such as MED, NEA and NWP, which accounted for the highest number of records and/or parasite species found. MED and NEA were also characterized by wider taxonomic range of parasites with respect to other regions. The geological history of MED and NEA probably led these regions to account for a peculiar and speciose cephalopod fauna, making them recognized hotspots and endemism-rich areas of cephalopod diversity37, so that the same geographic barriers that influenced the historical distribution of cephalopods throughout the Atlantic Ocean33 determined the nested pattern in their parasites. However, this anomaly was independent from nestedness in host assemblages among the three regions, which was indeed negligible, indicating that diversity centres of cephalopod parasites in the north-eastern and south-western portions of the Atlantic Ocean originated from evolutionary processes in parasitic taxa, rather than to dispersal and colonization patterns of their cephalopod hosts.Coherent biogeographic differentiation seems to underlay the overlap of β-diversity patterns between parasite and host assemblages, which were largely driven by species turnover in both cases. An exception to this general pattern was the Southeast Atlantic Ocean, where a large portion (60–70%) of the parasite pool was a subset of species from the Northeast and the Southwest Atlantic Ocean. Nestedness often originates from processes of ordered extinctions or colonizations along geographic gradients9). The taxonomic structure of the global parasite pool remained unchanged through time, conserving a taxonomic breadth and evenness which were undistinguishable from null expectations. More interestingly, we found that the dissimilarity in species composition of the global parasites pool over the last two centuries was almost completely driven by species turnover. As the number of studies increases and methods to detect parasites advance, one could expect that parasite species found in a given period should be, at least in part, a subset of those found in previous periods, so that nestedness should contribute substantially to the overall temporal β-diversity. This occurred, in fact, in the pool of cephalopod hosts sampled in the different periods, for which the turnover and nestedness-resultant components equally participated to the overall dissimilarity among periods. Instead, our counterintuitive findings indicated a reiterate replacement of parasite species over the last 200 years, in the absence of significant departures from expectation of taxonomic distinctness. Previous studies suggested that most of parasites species might be so rare that once they are found it could be very difficult to be encountered again in subsequent sampling38. The high temporal turnover in parasite assemblages and our estimate of the potential number of parasite species strongly supported this hypothesis, and clearly indicated that parasite diversity in cephalopods is far from being fully assessed.The number of parasites found in cephalopod hosts increased in time. This is not surprising since research efforts and the power to detect infections have improved due to new and more effective methodologies , which are likely to have rarely been recordedIllex, Loligo, Octopus, Sepia, and Todarodes) accounted for > 50% of parasite records. A possible explanation for this high parasite diversity may simply rely on the fact that they include species representing economically valuable resources exploited by fisheries and have received high research interest41. However, other factors related to species behaviour and spatial distribution could have played a role in determining the exceptional parasite richness.Five cephalopod genera , Trypanorhynchs  and for an assemblage of poorly known species of cestodes belonging to the order Phyllobothriidea 51, but also of different anisakid species, especially in the genus Anisakis. Squids belonging to the families Onychoteuthidae and Histiotheuthidae are the preferred prey items of beaked whales, which in turn represent the definitive hosts of the recently described species Anisakis ziphidarum Paggi, Nascetti, Webb, Mattiucci, Cianchi and Bullini, 1988 and Anisakis nascettii Mattiucci, Paoletti and Webb, 2009, suggesting that the life cycle of these species might be related to cephalopods as intermediate hosts20.In spite of their high diversity in terms of species richness, parasites of the genus 33 and of the overall marine biodiversity52. Polar regions were also poorly studied, and given the high peculiarity of parasite species pool that emerged analysing the record from the Southern Ocean, a substantial contribution to parasite diversity from research in these regions can be envisaged.Most oceans and seas, including potential hotspots of parasite diversity, merit further investigations to achieve a more comprehensive understanding of the magnitude of marine parasites diversity. The Western Central Pacific Ocean, for instance, was the least studied region deserving priority research investments, considering that the tropical indo-pacific zone represents a global centre of cephalopod diversityScolex and Phyllobothrium, but also for the Anisakis simplex species complex. For instance, Scolex pleuronectis, infecting several cephalopod species, is believed to represent a group of species belonging to different genera infecting a wide range of bony fish, cephalopods and gastropods worldwide53. Members of this taxon are further classified based on the number of loculi in the bothridia , contributing to increase confusion in taxonomic identifications. Future research should focus on molecular characterization of larval marine cestodes in order to increase the efforts in describing their life cycle throughout different hosts and resolve the taxonomic uncertainty about this group of marine parasites55. Problems related to taxonomic resolution also concern different protozoan parasites, particularly those belonging to the phyla Myzozoa, Ciliophora and Euglenozoa, among which the genus Aggregata (Myzozoa) is an emblematic example. In the full dataset we collected, only ten species of Aggregata were identified, each of them reported to infect a specific cephalopod species, while in the other cases these parasites were reported at the level of genus. Since marked host-specificity denotes the genus Aggregata, with preliminary molecular evidence also suggesting the occurrence of genetic differentiation of Aggregata species in different populations of their hosts9, detailed taxonomic analyses are needed to unveil the potential diversity hidden within this group of parasites.A major impediment for an exhaustive quantification of parasite diversity in cephalopods, and other marine organisms, is the scant knowledge on many speciose parasitic taxa. This is particularly true for cestode larvae belonging to the orders Tetraphyllidea and Onchoproteocephalidea, which are difficult to identify at species level based on the sole morphology, such as the parasites of the genus 58. In the marine realm, cephalopods are key ecological components of open sea and coastal ecosystems, with high socio-economic value for humans. Understanding the role of parasites in population dynamics, behaviour, and interspecific interactions of these molluscs is therefore essential26. Our overview of parasite diversity in cephalopods, by collecting and organizing parasite-host association records over the last centuries at a global scale, may represent a baseline step for future advances in this direction.For long time the ecological function of parasites has been overlooked, though their importance in maintaining biodiversity, regulating species coexistence, and modifying trophic interactions is undeniablewww.webofknowledge.com), the Natural History Museum Host-Parasite Database (www.nhm.ac.uk), and CAB Abstracts database (www.cabdirect.org). Additionally, the Biodiversity Heritage Library (www.biodiversitylibrary.org), and the Library of the Stazione Zoologica Anton Dohrn , were searched for older literature. Reports of infection of viral, bacterial and fungal agents were not considered. Databases were queried searching the keywords ‘cephalopod(s)’ and ‘parasite(s)’ in the topic field. The list of articles was then refined by selecting relevant ones based on their title and abstract. Finally, the selected articles were scanned to extract information on the parasite and host species found, location, and year of record. The full list of host-parasite associations and literature references is provided as Supplementary information (Appendix www.marinespecies.org) and species names revised if necessary. In very few cases , it was not possible to resolve invalid species names  represents the average taxonomic path length between two randomly chosen species in the taxonomic tree, whereas Variation in Taxonomic Distinctness (Λ+) reflects the unevenness of distribution of species within higher taxa and, therefore, the variance of these pair-wise path lengths. These indices have been applied to explore taxonomic diversity of several organisms in different environmental and geographic contexts around the world , including the study of host-parasite associations . Δ+ and Λ+ are independent from the number of species and virtually not influenced by sample size67, representing optimal tools when analysing incidence-based historical data .The Average Taxonomic Distinctness69.A taxonomic list, from species to phylum , was compiled with all parasite species of cephalopods recorded in the last two centuries from 1817 until 2017. Taxa identified at genus or coarser taxonomic level were considered as separate species. This list was used as a reference to calculate taxonomic distinctness indices and perform the associated statistical tests. All records of parasite species were organized in a data matrix including four subsets of data corresponding to 50-years periods from 1817 until 2017, so to have at least 50 records for each period. A second data matrix of parasite species (across the two centuries) was organized to include subsets of data corresponding to different geographic regions of recording and, namely, Northeast Atlantic (NEA), Northwest Atlantic (NWA), Western Central Atlantic (WCA), Eastern Central Atlantic (ECA), Southwest Atlantic (SWA), Southeast Atlantic (SEA), Western Indian (WI), Eastern Indian (EI), Northwest Pacific (NWP), Northeast Pacific (NEP), Western Central Pacific (WCP), Eastern Central Pacific (ECP), Southwest Pacific (SWP), Southeast Pacific (SEP), Southern (ANT) and Arctic (ARC) Ocean, Mediterranean (MED) and Baltic (BAL) Sea. This subdivision avoided excessive geographic fragmentation of records in the analysis, but it was still largely coherent with marine ecoregions proposed by Spalding et al.+ and Λ+ values for each period and in each region, which were tested to assess significant changes during the investigated time span or differences among regions in the taxonomic structure of parasite assemblages. To test the departure from expectations of indices calculated for regional subsets , the 95% ‘confidence funnel’ was obtained for Δ+ and Λ+60. This procedure generated 1,000 random subsets of m species from the reference list, calculated the index values and defined the corresponding 95% confidence interval. Randomizations were then repeated over a range of m values to obtain the confidence funnels against which testing the true values. Based on the same logic of funnels, a scatter plots of simulated Δ+ against the Λ+ values can be constructed, generating a combined 95% confidence ellipse for a specified number of species60. This bivariate approach was used to test the departure from expectations of both Δ+ and Λ+ values calculated on parasite assemblages from each historical period. For calculations of indices and 95% confidence intervals, the same step length  was used in weighting all distances between hierarchical taxonomic levels70.The reference list of parasites species and the two data matrices were used to calculate Δ+ and Λ+ of cephalopod assemblages in each region and tested their values following the same procedure described for parasites.Since the analysis highlighted significant departures from expectation of taxonomic distinctness of parasites for some regions see “” section71, to understand whether changes in species composition of parasites were mostly due to replacement or loss/gain of species. The same spatial and temporal patterns of dissimilarity were quantified also for hosts, and the Mantel test was used to assess correlations with those characterizing parasite assemblages. BAL, ARC, and CWP were excluded from this analysis due to the very low number of parasites recorded in these regions.Patterns of β-diversity of parasite assemblages in space and time were investigated by calculating the overall Jaccard dissimilarity among regions, and among the four periods. The overall β-diversity was partitioned into the two components of turnover and nestednessIllex, Loligo, Octopus, Sepia, and Todarodes, which altogether accounted for > 50% of parasite records. We used randomizations tests on Δ+ and Λ+ to assess whether parasites recorded for each of these genera belonged to specific taxonomic groups of parasites. We also calculated Jaccard dissimilarities among parasite assemblages characterizing each genus and quantified the contribution of turnover and nestedness to the overall dissimilarity.Due to their importance as commercial fishing targets and their relatively wide distribution, we carried out a specific analysis on five cephalopod genera, and namely 75. We used parasites associated to the 164 cephalopod species in the full record as a sample of parasite species richness in cephalopods. In order to estimate the expected number of parasite species that would be found in a larger sample corresponding to all extant cephalopods, we employed a non-parametric extrapolation method76. This procedure is based on the Bernoulli product model and allows extrapolation for sample-based incidence data, providing reliable upper and lower bound 95% confidence interval, particularly when sample size on which species richness is extrapolated is not very large with respect the size of the reference sample76, as in our case.Finally, the full record of parasite-host associations was used to extrapolate parasite total richness over the number of existing cephalopods, which account approximately 800 species77, R78 using the package ‘betapart’79, and the computer program EstimateS80.All analyses were done using the software package PRIMER v6All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Information 1.Supplementary Information 2.


Vaccines, has the main aim to increase international literature data on vaccine effectiveness and safety and on vaccination strategies in order to reduce vaccine hesitancy and improve vaccination coverage rates. The main topics included in the call for papers were vaccines administered to infants, adolescents, adults, elderly people, at-risk populations  and healthcare workers and strategies adopted to promote vaccination adherence among these categories. This Special Issue started from the assumption that, despite vaccination being universally recognized as one of the best strategies to increase duration and quality of life during the last centuries, vaccination coverage rates are often under the levels recommended to reduce circulation and to extinguish vaccine-preventable diseases. Vaccine hesitancy involves at least 15% of the general population, and healthcare workers also sometimes demonstrate doubts on vaccination effectiveness and safety. At the end of the six-month submission period, 16 articles (15 research article and one review) were accepted after the peer-review processes and published online.The Special Issue “Vaccination and Vaccine Effectiveness”, published in the journal  Vaccination is universally recognized as one of the best strategies to increase duration and quality of life during the last centuries . NeverthIn several countries, vaccination acceptance is low due to misconceptions or doubt about the effectiveness and safety of vaccines ,6. The vVaccines in June 2019 with the main aim of collecting international literature data on vaccine effectiveness and safety and on strategies that could contribute to reducing vaccine hesitancy and improving vaccination coverage rates [A Special Issue entitled “Vaccination and Vaccine Effectiveness” was launched by the journal Submissions of original articles, systematic reviews or meta-analyses, short communications and other types of article on vaccines administered to infants, adolescents, adults, elderly people, at-risk populations  and healthcare workers and strategies adopted to promote vaccination adherence among these categories were welcomed and encouraged for this Special Issue.Vaccines. In particular, 15 original articles (research articles) and one review article were published online in the final Special Issue.At the end of December 2019, 16 manuscripts were submitted and, after the peer-review process, accepted for publication in the Special Issue (SI) “Vaccination and Vaccine Effectiveness” of Several topics were addressed in the SI “Vaccination and Vaccine Effectiveness”, and all manuscripts published and available online in open-access form are reported in chronological publishing order in Pacenti et al. analyzed the seroprevalence of measles and vaccination coverage rates in the Veneto Region, Italy, from 2009 to 2017 among the general population and observed that measles outbreaks were common especially in areas with suboptimal vaccine uptake .At the University Hospital (UH) of Palermo, Italy, a quasi-experimental field trial was conducted from 2007 to 2019 in order to evaluate vaccination increase during consecutive influenza seasons among health care workers (HCWs) of the UH of Palermo after communicative and informative tailored strategies were adopted, and the impact of this increase on the reduction of working days lost due to acute sickness during influenza season .Piazza et al. conducted a retrospective cohort study on hospitalizations due to herpes zoster in Liguria Region, Italy, highlighting the considerable burden of this disease among elderly and frailpeople . Nicoli An observational study conducted by Cassimos et al. analyzed vaccination policies in 42 European countries, evidencing significant differences nowadays in vaccination programs for adults among the different countries considered in the study .Another study conducted in Sicily, Italy, evaluated the impact of HPV universal vaccine introduction into the vaccination schedule ten years ago, through the analysis of hospital discharge records. A significant decrease in hospital admissions for cervical cancer and other HPV-related diseases emerged after HPV vaccine introduction in Sicily, despite not satisfying vaccination coverage rates .Bechini and colleagues evidenced that more than half of internationally adopted children who visited Meyer Children’s University Hospital in Florence, Italy, between 2009 and 2018, if aged 15–18 years and originating from Africa, were not protected against MMRV . Mascia Among IAC with an unknown protective status against MMRV, as reported by Boccalini et al., a vaccination strategy based on serotesting results was the most advantageous approach, economically and clinically . MoreoveOne of the main challenges for public health, particularly for vaccinology, in the next decades is the contrast in vaccine hesitancy among the general population. In response to this phenomena, strong multidisciplinary alliances between health care professionals, providing evidence-based data on vaccine effectiveness and safety and on reduction of the burden of vaccine-preventable disease due to vaccinations offered, addressing vaccine hesitancy through innovative communication strategies and increasing opportunities to administer vaccines among the general population  are some of the possible strategies that should be considered by international and national health authorities and agencies ,27,28.


While modern biotechnology and, specifically, genetic modification are subject of debate in many parts of the world, an increasing number of countries in Sub-Sahara Africa are making important strides towards authorizing general releases of genetically modified (GM) crop varieties for use by farmers and agribusinesses. Obviously, the documented economic and environmental benefits from planting GM crops—based on a track record of over two decades—are a major driver in the decision-making process. Another key factor is the increasing alignment of biosafety regulatory policies with progressive agricultural and rural development policies in Africa, resulting in—compared to past experiences—greater emphasis on anticipated benefits rather than risks in biosafety regulatory reviews. In several cases, this has led to expedited reviews of GM crop release applications, either for confined field trials or general environmental release, taking experiences and data from other countries into account. Such regulatory approaches hold promise as the pipeline of relevant, pro-poor GM crop applications is expanding as are the opportunities provided by novel plant breeding techniques. This review article analyses the shifting policy context in select African economies, resulting in adoption of new agricultural technology, and novel regulatory approaches used in biosafety decision-making. Case studies will be presented for Ghana, Kenya, Malawi, Nigeria and Uganda to analyze challenges, distill lessons learned and to present general policy recommendations for emerging economies. As is the case generally when new technologies are introduced in society, there have been strong claims about the benefits and perceived adverse effects of agricultural biotechnology, specifically regarding genetically modified (GM) crops, and more recently regarding emerging techniques in plant breeding such as genome editing. Early concerns regarding genetic modification stimulated, from the 1980s onward, the creation of regulatory systems for food and feed safety and environmental risk assessment. In Africa, governments generally started the development of their national biosafety systems more recently and, as with other areas of safety regulation, the task has been difficult in terms of defining science-based regulations and enforcement. For most of them the Cartagena Protocol on Biosafety has set the starting point, as adopted in 2003 as a supplement to the Convention on Biological Diversity (CBD), and which seeks to address environmental impact from transboundary movement, management and safe use of genetically modified organisms (GMOs).The adoption of regulatory policies for biosafety and food/feed safety should be seen as part of a broader emerging international regime increasingly affecting the access and use of genetic resources for food and agriculture. This international regime affects scientific leeway and freedom-to-operate in a major way: The development and deployment of new agricultural technologies gets increasingly regulated, and often impeded, as access to essential research inputs  and the release of research outputs such as new crop varieties, is slowed down or halted by overly restrictive regulations. This phenomenon is described in further detail in “The culmination of these barriers renders some biological research untenable and can result in the abandonment of research projects before they even commence.”  crops; and,Increases in gross (in some cases net) margins due to the adoption of GM crops, or combinations of all the above.“Over the last 20 years, a large number of studies have been conducted, analyzing the effects of GM crop adoption on yield, pesticide use, farm profits, and other outcomes in different parts of the world. A meta-analysis has evaluated these existing studies, finding that GM crop adoption benefits farmers in most situations (…). On average, GM technology has increased crop yields by 22% and reduced chemical pesticide use by 37% (…). GM seeds are usually more expensive than conventional seeds, but the additional seed costs are compensated through savings in chemical pest control and higher revenues from crop sales. Average profit gains for adopting farmers are 68%.  benefits of biotechnology to improving food security and rural development, governments across Africa have taken steps to establish an enabling policy framework to support adoption of biotechnology including GM crops and derived products. A more detailed analysis of relevant policies and regulations is presented below. Examples of recent policy decisions regarding GM crops include:Approvals for general release and commercial variety registration for insect-resistant, GM cotton hybrids in Ethiopia (2018), Kenya (2019), Malawi (2019) and Nigeria (2018). While farmers in Ethiopia started planting GM cotton at limited scale in 2019, GM seed distribution in Kenya, Nigeria and Malawi will start in 2020;Approval for general release and submission for variety registration for insect-resistant, GM cowpea in Nigeria (2018). Next step in this process will involve the registration, by the National Variety Release Committee, of GM cowpea as a new commercial variety followed by seed distribution by local companies.In addition, with countries such as Ghana, Kenya and Uganda moving steadily from confined field trials (CFTs) towards general release applications, the setting for GM crop production in sub-Sahara Africa is rapidly changing. Until recently, only South Africa, Sudan and Burkina Faso had approved commercial production of GM crops. Right now, the regulatory pipeline is expanding and diversifying as illustrated by This increasing emphasis on agricultural biotechnology as a critical element in agricultural development policies is an important factor driving the expanding GM crop pipeline in Africa. Illustrative examples of such policies are presented below.As noted in a recent analysis by AGRA, the Alliance for a Green Revolution in Africa (2018), agriculture is key to Africa's future considering that the continent has most of the world's arable land, over half of the African population is employed in the sector, and it is the largest contributor to total gross domestic product (GDP). Yet, Africa is still producing too little food and agricultural value-added products . Product“[T]he drive to motivate farmers to adopt improved, certified seeds and fertilizers through a private-sector marketing framework, by raising incentives and complimentary service provisions on the usage of inputs, good agronomic practices, marketing of outputs.”  in order to enhance agricultural growth, alleviate poverty and improve quality of life . The MGD“The Green Alternative” aims to “build an agribusiness economy capable of delivering sustained prosperity by meeting domestic food security goals, generating exports, and supporting sustainable income and job growth” (Nigeria's Agriculture Promotion Policy (2016–2020) document, titled,  growth” . As the Agricultural policies in Uganda have been supportive towards exploiting the potential of GM crops since the development of the Plan for Modernization of Agriculture (PMA), some 20 years ago. The current overarching policy instrument, the Agriculture Sector Strategic Plan (ASSP) recognizes the need to enhance sustainable agricultural productivity and value addition using well-coordinated technological and service interventions . This poIn addition to the overall supportive policy initiatives sketched above, it is important to note that these countries have invested in establishing functional regulatory frameworks for GM crops, allowing decision makers to weigh potential benefits against potential adverse effects on the environment and human or animal health. A science-based and practical regulatory framework has become an important enabling factor for countries researching and adopting GM crops. In countries that are selected as case studies for this article, there has been progress in recent years in establishing functional national biosafety frameworks and growing expertise in GMO decision-making. An overview of key legal instruments and institutional setups is provided in Clear legal authority through comprehensive biosafety laws has still provided regulatory agencies with options to adopt flexible and innovative approaches to GM decision making. This has, for example, included decisions to (i) accept field trial data from neighbouring countries to expedite reviews of applications for confined field trials (CFTs); (ii) accept data from local GM CFTs and multi-location trials to shorten procedures for variety registration trials; (iii) accept food/feed safety dossiers and assessments from trading partners for accelerated safety decision-making. The following examples serve to illustrate these points.In Ghana, the National Biosafety Committee (NBC) approved multi-locational trials (MLTs) for insect-resistant GM cotton in 2012 after accepting data from confined field trials (CFTs) conducted previously in Burkina Faso. Considering the similar agro-ecological zones for cotton production in the two countries, the National Biosafety Committee (NBC) decided to grant an exemption for local CFTs and move to MLTs right away. These trials were put on hold in 2016 due to Burkina Faso's decision to terminate the commercial registration of GM cotton.Nigeria adopted the same principle—accepting data from other countries—and authorized country-wide MLTs for GM cotton prior to endorsing its general release and commercial variety registration in 2018.Kenya adopted fast-tracked protocols for variety registration trials involving GM crops that have gone through CFTs and MLTs, shortening the time required for DUS/VCUThese examples confirm that, increasingly, governments in Africa can adjust their regulatory decision-making processes based on accrued scientific evidence.While the above sketched progress in regulatory decision-making is encouraging, it is important to note that, still, most African countries are only slowly progressing in implementing functional regulatory frameworks. And, even in countries where recent progress has been achieved, there is considerable potential for backsliding. Many governments experience political opposition to GM crops and modern agriculture generally, and proposed biosafety legislation and regulations are conveniently associated with “opening the gateways” for the introduction of GMOs. Political opposition is in most cases fuelled by anti-GM activism, which has slowed down or halted the adoption of biosafety legislation . This has led to delays, inconclusive “conditional” release decisions and sometimes standstills in cases where these agencies lack familiarity with the biosafety regulatory review processes and tend to repeat safety reviews that were already completed. Early investment in establishing a coordinated, multi-agency framework is essential, coupled with policy consultations regarding harmonization of legal mandates.Post-release requirements: Towards the final stages of the regulatory review process, post-release requirements are being considered related to, among other things, product labeling, product liability, co-existence, monitoring and surveillance. These concepts are in many cases not yet implemented and tested in African countries, associated expertise is low, and enforcement will be problematic. In the early phases of regulatory framework definition in Africa, strict post-release requirements were usually formulated and only later it is realized that these will form an impractical impediment to technology deployment.de facto GMO import ban in Kenya  the continuation of a in Kenya , and  are at the forefront of incorporating NBTs in their research portfolio, in collaboration with national research organizations in sub-Sahara Africa. As a case in point, the International Center for Improvement of Maize and Wheat , the Kenya Agriculture and Livestock Research Organisation (KALRO) and Corteva  have joined hands to exploit the gene editing (CRISPR-Cas) technology to improve maize and wheat germplasm. A specific example where this technology will be employed is to address maize lethal necrosis (MLN), a devastating viral disease that has spread rapidly in many countries of East Africa since it was first detected in Kenya. CIMMYT identified a strong source of resistance against MLN, have fine-mapped it to a 1 MB region of chromosome 6, and expect to isolate the gene that confers resistance. Among the first targets for gene editing will be the parents of long-standing commercial hybrids in East Africa that were developed before the appearance of MLN and have since become susceptible to the disease. The International Institute of Tropical Agriculture (IITA), one of the CGIAR is on the forefront of applying NBT for improvement of banana for developing resistance to diseases . DetailsWhile most of the CRISPR/Cas9 based genome editing is reported in seed crops, recently it is also reported in vegetatively propagated crops like banana, cassava and potato . Genome Banana and cassava are among the important staple food and income generating crops for resource-poor farmers in Africa. Their contributions in the African diet are comparable to wheat, rice, maize, or potatoes in other parts of the world. The banana and plantain grown in Africa, is a starchy staple and the major source of carbohydrates to millions of poor people. Similarly, cassava is the most important primary food staple in several African countries. However, there are important biotic and abiotic constraints shattering the production of these important crops in Africa. Therefore, improvement of banana and cassava for economically important traits is critical to fulfill the food demand.Recently, CRISPR-based genome editing of banana has been demonstrated through knocking out the marker gene phytoene desaturase (PDS) leading to albino phenotype, however, the achieved mutation efficiency of 59% was quite low for practical application . FurtherMusa accuminata (A genome) or/and Musa balbisiana (B genome). Plantain (AAB), one of the economically important sub-groups of banana, contains one B genome. It is an important staple food crop in Africa. During BSV infection, multiple copies of eBSV sequences integrates at a single locus in the B genome of the host as direct, inverted and tandem repeats . The viral sequences of eBSV are integrated in the B genome derived from Musa balbisiana. Cultivated varieties of banana are polyploid  descended from wild progenitors  repeats . These pMusa balbisiana (BB) or several genotypes with at least one B genome have tolerance to biotic and abiotic stresses and good agronomic traits, still cannot be used as parents in breeding programs. The diploid progenitor Genome editing of cassava was established using the CRISPR/Cas9 technology for knocking out the Phytoene desaturase (MePDS) gene . This teFurther, researchers has attempted to apply this technology to develop enhance resistance to African cassava mosaic virus (ACMV), a geminivirus . HoweverCRISPR/Cas9-based genome editing can be coupled to genetic improvements in cassava for traits such as starch improvement and early flowering. In order to meet the increasing demand of food with limited or same resources, better and effective ways to produce food are required. As summarized above, one option is to utilize new breeding tools like genome editing for crop improvement. Currently, severe endeavors are underway to enhance yields of banana and cassava—among a range of other crops—through generating improved varieties with resistance/tolerance to biotic stresses.A major question to be addressed, from a regulatory perspective, will the products of NBTs be classified under the current definitions of genetic modification, or not? Over the last three decades, a patchwork of (draft) biosafety laws and regulations has emerged affecting the development and release of improved crops and resulting in trade issues when approvals are “asynchronous”Considering the above sketched situation, where differences in GMO regulation are exacerbated by specific challenges posed by NBTs, different countries are responding in different ways to the question of how applications of NBTs should be classified, as regulated GM material or not. The key point here is that specific applications, e.g., when genome editing is used to create a loss of function of a target gene, result in an event that contains no “foreign DNA”, i.e., no novel combination of genetic material and therefore cannot be distinguished from a product of conventional mutagenesis (which is commonly exempt from GM regulation).As early as 2013, the European Academies Science Advisory Council (EASAC), a body of national science academies of the EU Member States, argued that products of NBTs should not fall under GMO legislation when they do not contain “foreign DNA”. The EASAC advisors noted that in some cases the product cannot be distinguished from one generated by conventional techniques, and also argued that the new techniques allow much more precise and targeted changes compared with mutagenesis used in conventional breeding, where changes in the genome are induced by chemicals or radiation, creating multiple, unknown, and unintended mutations .novel combination of genetic material or not. Preliminary consultations are then followed by a final determination based on a full information package describing the gene editing procedure and resulting changes in the genomic sequences of the end product  to determine if a product developed using gene editing is a GMO or not. The prime decision-making factor here is if the product has a  product . So far,By contrast, in a major agricultural trading bloc like the EU there is still uncertainty as to how products from NBTs will be regulated. While EU bodies recognized the potential of NBTs early on, as evidenced by a range of studies and projects conducted with EU support, to date there has been no clear-cut policy decision or statement from the European Commission. So far its actions were limited to, among other things, requesting advisory notes from the EU Scientific Advisory Mechanism (SAM) and awaiting a legal opinion from the EU Court of Justice (CJEU). This wait-and-see approach has greatly complicated matters.“has been altered in a way that does not occur naturally by mating and or natural recombination”. This misunderstanding implies that organisms that have been edited but that can or do occur naturally, will have to follow the same European regulatory procedures as GMOs, including a detailed analysis of possible risks.On 25 July 2018, the CJEU advised that organisms obtained by new mutagenesis techniques are considered as GMOs, within the meaning of the EU's Directive 2001/18/EC on the release of GMOs into the environment (the “GMO Directive”), and that they are subject to the obligations laid down by the GMO Directive . Thus, tThe Court ruling has been widely debated since its publication, which will not be summarized here. A clear analysis of its implications was issued by the European Commission's Group of Scientific Advisors (2018). The Group concludes that:“[ … ] meeting the obligations of the GMO Directive implies cost- and labour-intensive pre-market evaluations and a long duration of the approval process, which are difficult and onerous to bear, particularly by small and medium enterprises. This may diminish incentives for investment, negatively affect research and innovation in this field, and limit the commercialisation of gene edited products.” , which would lead to a science-based guideline that would help implement the amended Act by including clear regulatory triggers for genome edited organisms.Rather than amending its biosafety act, in Kenya the National Biosafety Authority (NBA) opted to develop a guideline on genome editing. Guideline development was deemed to be a suitable approach as it allows for flexibility, in a rapidly evolving field, and consultations with scientists and regulatory agencies in the agricultural and environmental sectors. Following initial consultations, NBA organized a technical meeting in April 2019 analyzing advances in genome editing, and relevant regulations in Kenya as well as other countries. Currently, the authority is drafting a guideline that aspires to be practical and science-based, and which allows for case-by-case reviews and exemptions from biosafety review for products that do not have a novel genetic combination. Thus, the proposed approach for Kenya essentially follows those adopted in Argentina and other South American countries.It is expected that other countries that embrace agricultural biotechnology will soon follow Nigeria's and Kenya's lead and devise policy and regulatory approaches to NBTs and other emerging technologies.In this article, we have pointed to several important recent developments in sub-Sahara Africa regarding the regulation and adoption of GM crops, as well as the continuing political challenges that hamper further progress. Regulatory authorities in select case study countries have gone through a period of rapid capacity development, as shown by the increasing number and scope of GMO environmental releases including CFTs and general releases and have shown flexibility in their decision-making processes. A major driving factor in this process constitute the more progressive agricultural and development policies as formulated by national governments. Recent capacity development will provide a foundation for the formulation and implementation of science-based regulations for novel breeding techniques such as genome editing.While recent progress is encouraging, we fully acknowledge the fact that political challenges remain. A critical challenge involves the need to sustain the political will and current momentum that provides scientists with leeway to operate. While not discussed extensively in this article, it is generally recognized that controversies exist around the adoption of GM crops and that, despite a safety track record of over two decades, public opinion on this topic remains divided. These controversies are sometimes reflected in government decisions in sub-Sahara Africa, when decision-makers call for moratoriums on CFTs or bans on GM commodity imports. Such calls are influenced by activist groups who campaign against modern agricultural technologies generally, and against GMOs in particular. Recently, these campaigns have also resulted in court cases challenging biosafety decisions by national competent authorities. Despite this, progress in Africa continues but it involves a careful balancing act.Governments and development partners will have to continue investing in the development of knowledge, skills and capacities required to regulate and adopt GM crops; and, in due course, the products emanating from genome editing applications—as introduced in this article. Emerging best practices from, e.g., Argentina, provide critical guidance. Capacity development should include outreach and awareness initiatives to ensure public debates and policy consultations are well informed and incorporating the best available science.A special case is made here for enhanced regional and sub-regional collaboration. Increasingly, regulatory authorities in Africa are exchanging expertise and data regarding biosafety decisions, including the acceptance of data generated in neighboring countries. This collaboration would provide the basis for harmonization efforts in regional economic communities such as the Common Market for East and Southern Africa (COMESA), the Economic Community of West African States (ECOWAS) and the Southern African Development Community (SADC). Each of these regional bodies have initiated processes and guideline development towards sub-regional harmonization in the recent past but none have been adopted and implemented so far. A constructive development at the regional level involves the recent policy statements by the African Union (AU) regarding genome editing and gene drives for human health purposes, which have impacted discussions in AU member states. Such supportive statements will continue to be important to bolster the current momentum.All datasets generated for this study are included in the article/supplementary material. All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.Author JK was employed by the company Komen Consultancy.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The chicken industry of Pakistan is a major livestock sub-sector, playing a pivotal role in economic growth and rural development. This study aimed to characterize and map the structure of broiler and layer production systems, associated value chains, and chicken disease management in Pakistan. Qualitative data were collected in 23 key informant interviews and one focus group discussion on the types of production systems, inputs, outputs, value addition, market dynamics, and disease management. Quantitative data on proportions of commodity flows were also obtained. Value chain maps were generated to illustrate stakeholder groups and their linkages, as well as flows of birds and products. Thematic analysis was conducted to explain the functionality of the processes, governance, and disease management. Major chicken production systems were: (1) Environmentally controlled production (97–98%) and (2) Open-sided house production (2–3%). Broiler management systems were classified as (I) Independent broiler production; (II) Partially integrated broiler production; and (III) Fully integrated broiler production, accounting for 65–75, 15–20, and 10–15% of commercial broiler meat supply, respectively. The management systems for layers were classified as (I) Partially integrated layer production and (II) Independent layer production, accounting for 10 and 80–85% in the egg production, respectively. The share of backyard birds for meat and eggs was 10–15%. Independent, and integrated systems for chicken production could be categorized in terms of value chain management, dominance of actors, type of finished product and target customers involved. Integrated systems predominantly targeted high-income customers and used formal infrastructure. Numerous informal chains were identified in independent and some partially integrated systems, with middlemen playing a key role in the distribution of finished birds and eggs. Structural deficiencies in terms of poor farm management, lack of regulations for ensuring good farming practices and price fixing of products were key themes identified. Both private and public stakeholders were found to have essential roles in passive disease surveillance, strategy development and provision of health consultancies. This study provides a foundation for policy-makers and stakeholders to investigate disease transmission, its impact and control and the structural deficiencies identified could inform interventions to improve performance of the poultry sector in Pakistan. Chicken production is an important sub-sector of agriculture in Pakistan and plays a pivotal role in rural economic development. The estimated number of commercial chickens in the country in 2017 was 1,022 million birds with production of 17,083 million eggs and 1,270,000 tons of meat, providing direct and indirect employment to over 1.5 million people . The groThe initial rise 1960–1980) in growth of the chicken industry was promising but not sustained. Outbreaks of infectious diseases like hydropericardium syndrome, infectious bursal disease  and avia0 in growChicken meat accounts for 32.7% of the total meat production in Pakistan . It is aThe poultry industry in Pakistan is constantly evolving supported by government in form of tax reliefs, passing of the Punjab Poultry Production Act  and deveThere is a dearth of modern value chain tactics in the supply and marketing of chicken and its products  for mostMapping of production systems can provide an overview and understanding of the various production, harvesting, and distribution steps, types of actors and products involved along with their hierarchal position in value chains . FurtherChaudhry et al.  studied The main aims of this study were to characterize and map the commercial broiler and layer production systems and the value chains associated with these systems and to investigate options for chicken disease management and reporting in different production systems in Pakistan. The outcomes of this study generate information relevant for stakeholders, directly and indirectly involved in chicken production, who could be interested in identifying ways to improve value chain operations and design efficient disease control strategies.Broiler, layer, and backyard chicken production systems and their value chains were studied by collecting qualitative and quantitative data between October 2017 and December 2017 mainly by key informant interviews (KIIs) and one focus group discussion (FGD). Activities included (a) identifying the various value chain systems; (b) investigating their contribution to the total meat and egg production in the country; (c) mapping and describing the meat and egg value chains of different broiler and layer production systems. The latter included characterizing types of stakeholders, products and flows in the value chains, and identifying the services and measures taken for diseases like AI prevention and control.Punjab province was selected as the study area, because it accounts for the highest share in broiler and layer production in Pakistan with 608 million (63.25%) and 28.46 million (58.20%) out of 961.5 and 48.83 million broiler and layer birds, respectively  industry and food systems were identified and approached for scoping interviews. The aim of these interviews was to gain a high-level overview of the chicken production, its structure, types, trading systems, and disease control systems. Additionally, these interviews were used to identify the major key informants (KI) and stakeholders involved in the chicken value chain, who could then be subsequently contacted for more detailed interviews.During the scoping interviews, participants were asked to: (1) describe the different production systems in terms of purpose, species, management, husbandry (including housing), and number of birds; (2) estimate the proportions of different poultry production systems in Pakistan; (3) provide an overview of the value chain nodes in the chicken production systems including identification of stakeholders and key markets or infrastructures.n = 1) with nine independent broiler farmers was initially conducted, but this approach was replaced by face-to-face interviews with key informants (n = 23), as the FGD was perceived to be inefficient and unproductive due to cultural dynamics.Following the scoping study, the KIs or stakeholders identified by the participants of the scoping interviews, were interviewed using semi-structured interviews . A FGD (During the FGD and KIIs respondents were asked to describe the following: (1) flock size and type of birds; (2) sources of inputs ; (3) types and distribution of outputs ; (4) types of people involved ; (5) flows of inputs and outputs and their association with one another; (6) amount of different outputs obtained; and (7) institutions and people involved in disease reporting, control and management.Interviewees were asked open-ended questions . Various prompts were used to explore and clarify details on activities, people involved and product flows. The participants were asked to describe and discuss people, inputs, outputs, flows, and quantities in the system. During this process, the interviewer drafted flow charts that participants could clarify and amend. This iterative process was followed to create a preliminary map of the system by the interviewer; it was subsequently checked and approved by the interviewee. All interviews were conducted in Urdu language by the principal author and were audio recorded. Additionally, summary notes were taken of all discussions held. The complete question guide is available in Through careful listening of the audio recordings the data were translated and transcribed. The notes and flow charts taken during the interviews and the FGD were then added to the transcribed recordings in a Microsoft Word document which allowed initial familiarization with data and preliminary structuring of information.Analysis was done in two parts: first, a mapping analysis was performed to assist in the creation of flowchart diagrams, building on the drafts from the KIIs. This step allowed the creation of mapping profiles for different sections of the broiler and layer systems. These showed the type of people involved, flow of inputs, outputs and other chain characteristics that are key components of broiler and layer production systems. Where possible, proportions, or sizes were indicated as integers and with arrows of variable widths, according to the magnitude of the flows.Secondly, thematic analysis was performed to identify meaningful themes that would provide understanding of the processes, governance, and interactions within the chains. Data were imported from Word into NVivo software  and coded and arranged on the basis of similarity of information in the codes. Subsequently, the various themes were identified based upon codes that described an activity or characteristic of a value chain node. This thematic analysis was used to refine the mapping profile generated in the first step. Every time an interaction, stakeholder or activity was mentioned as associated to a particular chain, this was added to the mapping flow-chart diagram. Based on the broad topics asked during interviews, key themes of governance in the form of dominance, management, health provision, and identification of structural deficiencies in the poultry sector were identified and coded. All categories and themes were proof-read by co-authors as a quality check to avoid any gaps in theme identification and categorization.Figures 2, 6). Fully integrated producers were those where one single company managed the value chain, from grandparent production to finished chicken products sold to retailers or consumers. Partially integrated producers included companies that managed parent stock/breeder, finishing of broilers or layers, with varying control of the distribution of products or finished birds (but no grandparent stock). Independent farmers were described as broiler and layer growers who practiced rearing of day-old chicks (DOCs) to the level of finished birds in the case of broilers, and to egg production in the case of layers.Respondents described that over 75–80% of chicken meat and egg production in Pakistan was commercial in environmentally controlled intensive systems, while backyard birds accounted for 20–25%. Keeping chickens in the backyard was described as subsistence to meet household meat and egg consumption using free range systems. Three production systems for broilers and two for layers were identified . These sCommercial broiler farming management is divided into environmentally controlled and open-sided house systems. Major broiler producing areas in Pakistan were reported to be in Punjab, in particular in the districts of central and north Punjab .Major production system with their value chains are shown in Key informants reported that Arbor acres, Hubbard, Cobb, and Ross are the broiler breeds most often used in Pakistan. It was perceived that Hubbard breed was more popular in the past, but has lately been replaced by Cobb accounting for 50% of total broiler production. Hubbard, Ross and Arbor acres were estimated to represent 25, 15, and 10%, respectively. This shift from Hubbard to Cobb was believed to be due to a change in farmers' and consumers' preferences. The major reasons included “better FCR,” “comparable quality of meat,” and “increased amount of breast meat per bird.” Breast meat was reported to be directly related to profit gained, and hence preferred.These systems were characterized by single ownership of the entire value chain . There wCentral management of these companies' broiler growers was reported to ensure good farm practices and quality of the finished product, through maintaining environmentally controlled systems and strict biosecurity protocols. Company-operated vehicles were said to transport finished birds from their grower farms to processing plants that are mainly located on the outskirts of Lahore city, with the capacity to process a maximum of 50,000–60,000 birds per day. It was also reported that these companies are Halal (ISO 9001) and international food safety management system (ISO 22000) certified and operate using a hazard analysis critical control point system. Chickens were processed into frozen carcasses, meat cuts and ready to cook products (98–99%), with 1–2% by-products that included shanks with claws, feathers, intestines and blood. Shanks with claws were exported to China while other by-products were sold to commercial rendering plants. Finished products were supplied to the company's own outlets and to independent retail grocery shops, supermarkets, restaurant chains, and various institutions like hotels and clubs throughout Pakistan via company-operated refrigerated vehicles.Interviewees explained that the processed products were mostly used domestically in the country, although frozen carcasses and ready-to-cook products were also exported to Middle Eastern countries like Qatar, Abu Dhabi and Bahrain. Export was described to be the only part of the FIBP value chain moderated by middlemen. In case of high demand of processed chicken from target customers, these companies were reported to purchase broiler birds from independent broiler farmers. Such deals were described to be devoid of any middlemen involvement and required strict bird health criteria to complete the purchase, which include birds being negative for Salmonella, Mycoplasma infection, and free from antibiotic residues.Operations of PIBP are shown in The PIBP broiler value chain was found to include either informal middleman-governed distribution chains, or formal company-operated distribution chains. The respondents explained that the majority of finished broilers (80%) were sold as live birds to designated brokers who have contracts with the companies. These brokers have additional contracts with traders to collect finished birds from the farms and supply them to live bird markets  or independent poultry processing plants. The remaining 20% of the finished broilers produced were said to undergo processing to finished broiler products via company-operated processing plants followed by transport via company-designated refrigerated vehicles to local restaurants, grocery outlets, supermarkets, and the companies' own outlets in the country.The PIBP as explained by the interviewees were in transition from independent broiler farms toward a fully integrated system. The reasons reported for this transition were the “unstable live broiler market,” “price fluctuations due to seasons and festivals,” and to “bypass middlemen” dependency in the distribution chains.It was estimated that independent broiler farming  is a majFarm level decisions in IBP such as selecting breed of broilers, sourcing feed, and vaccination were reported to be made by either the farmer, farm manager or farm supervisor. These decisions were reported to be autonomously made without being influenced by any FIBP or PIBP, despite them being major providers of DOCs and feed to independent farmers.Independent broiler production was further categorized into large and small scale depending on total housing capacity. Small scale broiler farmers were described to have capacity to house between 30,000 and 0.5 million broiler DOCs, while large scale broiler farmers could house between 0.5 and 1.5 million birds; very few farmers were reported to have capacity for >1.5 million broiler birds. To reduce the cost of production and increase efficiency, it was reported that 85–90% of IBP is within environmentally controlled systems. The major input costs as perceived by the farmers were for feed (60–65%) and DOCs (25–35%), followed by vaccination and medicine (12–15%). Feed and DOCs were provided directly to the farm level by the independent feed millers and breeders, FIBP, PIBP via company-operated vehicles without the involvement of any middlemen. Large scale broiler farmers tended to produce their own feed with extra feed purchased commercially if needed. In contrast, small scale broiler farmers were reported to depend on commercially available feed, mostly supplied on a credit basis. The majority of DOCs (80%) supplied to these independent farmers is provided by 10–12 companies (FIBP or PIBP), and their distribution offices were reported to be located around the major poultry producing areas, such as central and north Punjab. These companies have 80% of the country's parent stock flocks, while the remaining 20% parent stock is distributed among small and large independent broiler breeder companies.Numerous chains with middlemen, live bird market and wholesale markets were found for the sale of finished broiler within IBP , 5. ThesIndependent broiler farming (IBF) was described as a major contributor to Pakistan's broiler production. Live bird markets serving this production system dominate; they link brokers, traders and suppliers in the sale of finished birds . FarmersTo ensure halal slaughtering and respond to consumer preference for freshly slaughtered meat at cheaper rates, wet chicken markets in the form of retail outlets  were described to be more common than other sources. These stalls were distributed all over the country in and around residential areas, serving freshly slaughtered halal meat daily based on demand. They were reported as the major provider of chicken meat to low- and middle income customers. Conversely, the FIBP and PIBP supplying a wide range of value-added products tend to target high-income consumers by selling these processed products at a higher price in more formal settings and ensuring that food safety standards are met.Respondents stated that layer production is less profitable than the broiler production due to; a longer production cycle creating seasonal placements (February–March) of layer DOCs in rearing houses, high feed costs, and a lower probability (33%) of getting female chicks out of hatched eggs. A total of 40,000–50,000 layer breeders were present in Pakistan; they were distributed among 7–8 layer breeder companies and big layer production farms. Layer farming was mostly practiced as independent farming or as partially integrated layer production. Processing of table eggs was not reported to be commonly practiced and only one independent company throughout the analysis was reported to process eggs into egg powder and packed liquid eggs.The major layer breeds in Pakistan were Hy-Line , Babcock, Lohman , Novogen , H&N international  and Hendrix genetics . LsL light was believed to be the most popular breed in Pakistan due to “high egg production efficiency” and its “fitness for the cage system” because of its light weight. Average peak production of commercial layers in Pakistan was described to be reached at the age of 26–29th weeks with an average number of 320 eggs produced per bird per unit production cycle. Major layer producing areas reported in Pakistan included districts of central Punjab , north Punjab  and south of Sindh province (Karachi and Hyderabad) .; there were no reports of production and breeding of layers' grandparents in the country. The layer parent stock is imported from United States and Europe, and kept either by partially integrated companies or by independent breeders. Companies were designated as partially integrated due to their absence of keeping layer grandparent and partial ownership of distribution chains (40%). All major inputs like feed and DOCs were reported to be provided by company-owned feed mills and hatcheries.The interviews revealed that no fully integrated layer production system exists in Pakistan ; there wThe market share of egg production of PILP in Pakistan was estimated to be around 10–15% . Only twThe partially-integrated layer companies had their own egg distribution chains, called “branded sale” (40%), while the rest of supply was moderated by middlemen (60%). Branded sale was reported to include grading and packing eggs before distributing it to hotels, clubs, restaurants, supermarkets, retail shops, and company owned outlets via company-operated vehicles. Moreover, such companies were also reported to add value to eggs by Omega 3 enrichment and deeply-pigmented yolk in line with consumer preferences. These value-added eggs were only 10% of all eggs produced by partially integrated companies.The market share of ILP was about 80–85% of the total egg production in Pakistan . Only a In open-sided house based farming layer birds were kept on the floor during the production phase with manual egg collection performed 4–5 times a day, in contrast to cage production where all eggs were automatically collected once a day, thereby reducing labor costs. In ILP, 60% of the eggs were reported to be traded through open markets dominated by brokers, traders and “mobilers” . MobilerBackyard production in Pakistan was defined as keeping 2–3 birds in the backyard of a house for recreation or domestic use. Backyard production was predominantly found in remote areas of Pakistan where it is difficult to maintain a continuous supply of inputs. The backyard birds were reported to be mostly kept in rural areas to meet household needs of eggs and meat with surplus eggs and spent birds sold to hawkers. Hawkers were said to sell these birds to local markets/retailers and wholesale markets of big cities like Lahore. The breeds that were kept for backyard farming included Desi, Fayoumi, Rhode Island Red, Naked neck, and their crosses. The housing system was free range and feed sources included scavenging and kitchen by-products. Major areas of backyard farming included rural areas of Chakwal, Mianwali, Bhakkar, and Dera Ghazi Khan Districts of Punjab. As the backyard farming was scattered through secluded rural areas of Pakistan, backyard farmers were reported to have only access to health facilities provided by rural government veterinary or para-veterinary staff.Interprovincial unregulated transport of chicken was evident in the study. The production surplus in Punjab province, along with high production prices in other provinces were described as major factors that caused movements of birds from Punjab to other provinces. Some participants explained that traders from other provinces acquired birds from Punjab if there was enough profit margin left after deducting transportation cost and weight loss during transport. The price difference of broiler meat between Punjab and other provinces was estimated at 40 PKR/Kg (0.26 USD/Kg) live weight and farmers reported that traders from other provinces traveled a distance of 900–1000 Km toward central Punjab to purchase live birds at a cheaper rate.Respondents described cross-border trade with Afghanistan for exporting finished broilers, spent hens and table eggs from Pakistan. Due to a lack of import standards in Afghanistan these exports were described to be free of any safety checks and quarantine procedures. This trade was reported to be moderated by the traders on either side of the border based on credit or cash depending on the type of agreement. However, the respondents showed concerns about “cash recovery” in this trade.Commercial broiler and layer producers were located close to urban settlements to allow easy access to the markets. A rapid increase in using processed meat and ready-to-cook chicken products by consumers was reported. This was mainly due to increased number of large- and small-scale slaughterhouses around the major poultry producing areas of Punjab province. Improved meat processing technologies, good hygienic practices, and strong marketing tactics, with electronic and print media used to increase consumer awareness about the safety and hygienic food, were the major reasons reported for increased consumption of chicken products. Such marketing campaigns were moderated by meat and egg processors mostly targeted at medium-high income customers as low-income groups cannot afford to pay 50–100 PKR (0.5–1 USD) extra for the same weight of chicken that they could easily get from informal chicken stalls. The situation was described to be similar for egg marketing. Respondents also hypothesized that with increasing demand of ready-to-eat products and ease in household handling of processed meat these markets could overtake wet markets in the future.The availability of poultry health services varied among different chicken production systems. Two main types of stakeholders  were repMedicines and vaccines were reported to be purchased directly from regional distributors or veterinary pharmacies, and veterinarians were found to be involved in their marketing and sale. These veterinarians also provided free health consultancies to independent chicken farmers. Only one company (PIBP) was found to operate their own pharmaceutical units producing medicine including antibiotics for their own farming business and for commercial sale. In case of any notifiable disease outbreak, especially high pathogenic AI at the district level, it was the responsibility of the local government veterinary officer to inform the assistant disease investigation officer, who further informed the divisional disease investigation and control officer followed by provincial and federal reference laboratories. The Pakistan Poultry Association was found to be the major organization actively working as a link between government and poultry industry to address the issues of the poultry farming community at federal level. At provincial and federal level, strategies were developed and updated in consensus with Pakistan Poultry Association under the umbrella of Poultry Production Act, 2016, to devise and disseminate information on disease control and interventions. This included restriction of animal movement, adoption of strict control measures and increased surveillance in the affected areas. However, for low pathogenic AI H9N2 virus no special reporting system was stated during the interviews. In FIBP, PIBP and independent large-scale farming, internal disease reporting systems, including H9N2 AI infection, were reported that did not involve government veterinarians. Most of the reported H9N2 AI outbreaks in Pakistan happened between the months of March–April and October–November. The farmers were aware of such seasonal outbreaks and “mentally prepared” for losses during H9N2 outbreak months.Respondents in all integrated and large-scale independent chicken production systems described burying or burning diseased and dead birds. Most of the small-scale independent farmers reported disposing of birds on landfills and in canals located close to their farms. The large-scale farmers showed concerns that such improper disposal and lack of regulations from government on disposal practices were major reasons for repeated outbreaks of H9N2 AI. Structural deficiencies as reported by the respondents during interviews are presented in The aims of this study were to characterize and map chicken production systems, associated value chains and to explore the options for chicken disease control in Pakistan. To the authors' knowledge, this is the first study to provide a detailed characterization of the chicken production systems and their value chains in Pakistan.The mapping of the chicken production industry identified important differences in production types, chain structures, and marketing of finished products. The chain structure varied in terms of length and intricacy across profiles. Short chains were present in FIBP and some PIBP setups, while long chains were mainly found in independent production systems, where middlemen like brokers, traders, suppliers and mobilers were not only involved in distribution of products, but also in price control. While price control may be beneficial for single actors or a group of actors, it increases transaction costs and contributes to inefficiency within poultry value chains; this is in line with similar findings in other low and middle income countries –20. It wPeople's perceptions about meat obtained from chicken stalls as fresh, halal, easy to access and cheap are factors causing wet markets to dominate. These findings are consistent with those of Karthikeyan and Nedunchezhian  in IndiaThe chicken market in Pakistan (broilers and spent hens) is predominantly regarded as a live bird market and large independent and partially-integrated farmers were found to be involved in the export of live birds to Afghanistan–as previously reported . FarmersIntegrated companies in Pakistan were found to be involved in the export of processed and packed chicken meat and egg products including frozen carcasses, ready to cook items and value added meat and eggs; a practice reported earlier in India . HoweverDOCs and feed in Pakistan are mainly supplied by integrated production systems, which is in accordance with findings from Kenya and Pakistan where dominance of large companies in supplying DOCs has been described , 18, 23.Poor farm management practices were reported in the study that could play a role in AI outbreaks on farms , 30. StrPrivate and public sectors were equally important in controlling poultry diseases including AI at country and farm level. These stakeholders could be targeted to inform policy making and develop robust approaches for disease control . The stuThe current study has some limitations. The qualitative nature of the study means that a limited number of participants were interviewed. However, participants were carefully selected because of their extensive knowledge of (parts of) the poultry value chains and to ensure a broad representation of diverse stakeholders. Their views, although believed to be a good approximation of the chains structure and its working, may present some bias. We included participants from large corporate level to small backyard farmers, experienced consultants to farm veterinarians, and large and small poultry traders that helped to cover the major aspects of the poultry sector from wet to processed markets, local, and export markets and disease control options in various poultry settlements. Because of challenges related to social and cultural norms, only one FGD was possible and the remaining data were collected through KIIs conducted face to face. It provided a chance for participants to express their opinions freely. Proportions like market shares, country level shares obtained during data collection were merely based on approximations and personal perceptions of respondents and their average is represented in the results. By-product chains were not explored in depth, but a brief description of various by-products was included.The mapping gives an in-depth understanding of the structure of the chicken value chains in Pakistan thereby providing a basis for epidemiological disease modeling. Such modeling could help to identify critical control points for interventions toward safe and sustainable food. Identification of actors across various levels of the value chain can be used in further research to investigate personal beliefs and behaviors in relation to control measures. Finally, information on the linkages and processes in these chains provide a starting point for detailed investigation of transaction costs.Detailed value chain maps and information on integrated, independent, and backyard production were used to characterize the chicken industry in Pakistan, and to highlight structural differences between broiler and layer production systems. The analyses revealed the dominance of specific stakeholders, actors and markets in supplying chicken and its products throughout the country. Processed markets were mainly captured by FIBP, PIBP, or PILC where the role of middlemen was negligible, while the wet market was dominated by independent farmers where middlemen influenced the pricing of goods and supplied live birds and eggs to chicken stalls and retail shops. Lack of efficient government policies on price control and farm biosecurity were reported to lead to price fluctuations and inefficient disposal of dead birds. The current study provides baseline information on chicken value chains in Pakistan and identifies factors causing disruptions in the operations of this sector, along with aspects that influence prices. It can be used as a basis for economic impact assessment of chicken diseases and the calculation of economic efficiency of vaccines in different production systems. Stakeholders identified could be targeted for devising policies and novel interventions for efficient control of diseases in the industry.The datasets generated for this study are available on request from the corresponding author.Ethical approval was sought from and granted by the Social Sciences Research Ethical Review Board (SSRERB) of the Royal Veterinary College, UK (project reference: URN SR2017-1303). Prior to data collection, informed consent was obtained from all participants for participation in the study, audio recording and photography. The participants provided their written consent to participate in the study.HA collected, analyzed data, and drafted and revised manuscript. BH and PA were directly involved in developing study design, data analysis, writing of manuscript, and critically reviewed all manuscript drafts. MI contributed toward initial project planning and grant winning , technical support in data collection, and reviewed drafts of manuscript. TY contributed toward provision of logistical and technical support while collecting data in Pakistan and reviewed drafts of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.