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Reconstruction of wildlife distribution based on poorly-sampled data

Reconstruction of wildlife distribution based on poorly-sampled data


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cross-posted to Signal Processing, Cross Validated, and World Building This Site

Hi, I thought I'd also put this here in case there are any field biologists with ideas on the matter.

Problem: After reading a series of fantasy novels, I noticed that the biosphere in that world made no sense. To clarify, this is a world where despite magical occurrences, the world itself is almost entirely non-magical. 'Alternate history magical realism,' perhaps. i.e., unlike, for example, Harry Potter, in which almost all plant and animal species mentioned are fictitious and magical, this series uses real flora and fauna. This allows me to extract information about the fictional world's environment based on the distribution of these animals, by assuming that similar animals will live in similar climates on Earth and in the fictional world.

Ignoring the likelihood that the original author did not put enough thought into worldbuilding to make this a necessarily reasonable endeavor, my idea for how to proceed was as follows:

As maps exist of the fictional world, and the path of the characters can be plotted, I hoped to mark every mention of a specific plant or animal in the text, along with the location of the characters when it occurred, and from this reconstruct a plausible distribution for each species. I've created a theoretical example (in photoshop), for illustration:

where the red dotted line represents the paths of various characters, the orange, green, and blue splotches represent the true distribution of the species; the stars, triangles, and circles represent the locations at which a species is mentioned; and the brown, green, and blue lines represent the reconstructed contours of the distribution.

Is there a method to do such a reconstruction? It sounds a bit like a Monte Carlo analysis, but I figured I should check… (It also sounds rather like the magical programs detective shows use to plot serial killers' locations)

Note: It should be clear from the problem statement that just because a species is not mentioned at a specific location does not mean that it does not exist there. i.e., a sample at a specific location returning only 'A' - 'Bill and Jeff saw a lemur.' - does not exclude the possibility of 'B' and 'C' also at that location, but not sampled. Just because the text may specifically say that Bill and Jeff saw a lemur, and doesn't mention any other flora or fauna doesn't mean we should assume that they are in a universe devoid of anything but the occasional lemur.

Final Thoughts: Ideally, the analysis method would further:

  • take into account the coverage of the paths, and not assume that (in the example above) nothing exists in Mexico or northern Canada, just because there are no samples taken there. Remember that samples can only be taken along paths.
  • take into account edges, in this case coastlines. If A, B, and C are land animals, it does not make sense that a reconstruction of their distribution would include water, even if their range surrounds a lake or something.

Sorry for the long-winded explanation. Any thoughts?


The problem of how to infer species distributions from scattered species occurrences is common in ecology, and there exists a number of methods to construct distribution maps. As a start, you should have a look at Species Distribution Models (SDMs) using regressions models or Maxent, and the paper by Elith et al (2009) is a good starting point and a standard reference. SDMs using maxent is now a common approach, which integrates species occurrences as point data along with environmental layers (e.g. temperature, moisture and topography) to predict species distribution maps, and this can also include absence data or "pseudo-absence" data (randomly sampled data from a region of interest). The maxent software is described and can be downloaded here: http://www.cs.princeton.edu/~schapire/maxent/ A common criticism against distributions produced by Maxent is however that they ignore e.g. species interactions, and they only considers the species occurences and the environmental variables that has been included in the model.

In your "Note", you touch upon the issue of detectability, which is an important issue that has received much attention recently. The problem is largest when you only have presence data, and to have real presence/absence data is preferable. Even if you don't have real absenses (the species has been searched for but not found), an estimate of sampling effort in different areas is still very useful, since this means that you can at least evaluate whether absenses is due to "real" absense or lack of sampling. In your case, the movement paths of characters could be used as a measure of spatial "sampling effort". The main issue with detectability in studies of distribution of species trends is if there is trends or bias in detectability, which means that apparent changes over time or patterns in distribution might be due to differences in detectability and not real differences between areas or over time. This could for instance be the case if observers are more likely to spot a species in one type of habitat (open savannah) then in another type of habitat (closed forest). Useful starting points for issues of detectability are Dorazio (2014) (technical though) and Kery et al (2010).


Reconstruction of wildlife distribution based on poorly-sampled data - Biology

eDNA is driving rapid advances in ecology, evolution, and conservation.

eDNA provides mechanistic insights into ecological and evolutionary processes.

Foremost among these is an improved ability to explore ecosystem-level processes.

We examine current frontiers of eDNA, outlining key aspects requiring improvement.

We suggest future developments and priorities for eDNA research.

Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through the use of time-serial samples and unprecedented sensitivity for detecting rare or difficult-to-sample taxa. Although technical challenges remain, here we examine the current frontiers of eDNA, outline key aspects requiring improvement, and suggest future developments and innovations for research.


Agent-based simulation for reconstructing social structure by observing collective movements with special reference to single-file movement

Understanding social organization is fundamental for the analysis of animal societies. In this study, animal single-file movement data —– serialized order movements generated by simple bottom-up rules of collective movements —– are informative and effective observations for the reconstruction of animal social structures using agent-based models. For simulation, artificial 2-dimensional spatial distributions were prepared with the simple assumption of clustered structures of a group. Animals in the group are either independent or dependent agents. Independent agents distribute spatially independently each one another, while dependent agents distribute depending on the distribution of independent agents. Artificial agent spatial distributions aim to represent clustered structures of agent locations —– a coupling of “core” or “keystone” subjects and “subordinate” or “follower” subjects. Collective movements were simulated following two simple rules, 1) initiators of the movement are randomly chosen, and 2) the next moving agent is always the nearest neighbor of the last moving agents, generating “single-file movement” data. Finally, social networks were visualized, and clustered structures reconstructed using a recent major social network analysis (SNA) algorithm, the Louvain algorithm, for rapid unfolding of communities in large networks. Simulations revealed possible reconstruction of clustered social structures using relatively minor observations of single-file movement, suggesting possible application of single-file movement observations for SNA use in field investigations of wild animals.

Significance statements

An agent-based model was developed to test if animal single-file movement is informative for the reconstruction of clustered social structures.

Single-file movement data sets were generated with simple bottom-up rules of collective movements.

Simulations showed possible reconstruction of clustered structures for most combinations of parameter settings

Latent structures of clustered animal groups might be identified by collective movement observations, and single-file movement may be practically applied to wildlife investigations.


Introduction

Tuberculosis (TB) is the leading infectious killer in the world and approximately 10 million new cases are reported annually. In 2017, 1.6 million people died of TB and over 95% of these deaths occurred in low and middle-income countries (World Health Organization [WHO], 2018). The disease is strongly linked to poverty, with its prevalence following a socioeconomic gradient within and among countries (Lönnroth et al., 2009). In addition, there is a significant, and often neglected, contributor to the global disease burden, which is the zoonotic transmission of bovine TB to humans (Olea-Popelka et al., 2017). The WHO (World Health Organization) estimated that 142,000 new cases and 12,500 deaths occurred due to zoonotic TB in 2017 (World Health Organization [WHO], 2018), numbers that are likely underestimated due to lack of routine surveillance data from most countries (Olea-Popelka et al., 2017). People with zoonotic TB face arduous challenges most strains of the etiologic agent are resistant to pyrazinamide (Konno et al., 1967 Scorpio and Zhang, 1996 Loiseau et al., 2019), one of the first-line drugs used in TB treatment, and a possible association with extra-pulmonary disease (Dürr et al., 2013) often delays diagnostics and treatment initiation (World Health Organization [WHO] et al., 2017). In addition, bovine TB results in severe economic losses for livestock producers worldwide, respecting no borders and repeatedly affecting animal conservation efforts due to the establishment of wildlife reservoirs or spillover events from cattle to associated animal populations (Ayele et al., 2004 De Kantor and Ritacco, 2006 Godfray et al., 2013 Miller and Sweeney, 2013 Palmer, 2013 Nugent et al., 2015a, b). In order to eradicate TB by 2,030 as part of the United Nations (UN) Sustainable Development Goals, it is imperative that future prevention and control strategies focus on all forms of TB in humans, including its interface with animals.

Human and animal TB are caused by members of the Mycobacterium tuberculosis Complex (MTBC). The MTBC is a clonal bacterial group composed of 12 species or ecotypes with variable virulence and host tropism (Galagan, 2014). Mycobacterium tuberculosis stricto sensu is the main responsible for the TB numbers and is adapted to human hosts (Brites and Gagneux, 2015 Malone and Gordon, 2017). On the other hand, Mycobacterium bovis, the causative agent of bovine TB, has a broader host range and is able to infect and cause disease in multiple host species, including humans, with variable populational persistence (Malone and Gordon, 2017). MTBC members have clonally evolved from a common ancestor with the tuberculous bacteria Mycobacterium canettii (Supply et al., 2013), and alignable regions of MTBC genomes are over 99.95% identical, with horizontal gene transfer and large recombination events considered absent (Hirsh et al., 2004 Gagneux and Small, 2007 Galagan, 2014). These pathogens have solely evolved through single nucleotide polymorphisms (SNPs), indels, deletions of up to 26 Kb, duplication of few paralogous genes families, and insertion sequences (IS), which translated into a phenotypic array of host tropism and virulence variations (Brosch et al., 2002 Gagneux and Small, 2007 Lazzarini et al., 2007 Galagan, 2014 Brites et al., 2018).

Using whole-genome, SNP-based phylogenetic analyses, human-adapted MTBC have been classified into 7 lineages, with M. tuberculosis accounting for L1 to L4 and L7, and Mycobacterium africanum comprising L5 and L6 (Coscolla and Gagneux, 2014). Each human-adapted MTBC lineage is associated with specific global geographical locations, and lineage-associated variations in virulence, transmission capacity and in the propensity to acquire drug resistance have been reported (de Jong et al., 2010 Portevin et al., 2011, 2014 Gagneux, 2012 Sarkar et al., 2012 Coscolla and Gagneux, 2014). Thus, regional prevalence of specific lineages or sub-lineages have consequences for the epidemiology of TB worldwide. A similar attempt to classify M. bovis into different genetic groups was made prior to the large-scale availability of whole-genome sequences and started with the identification of clonal complexes (CCs). Accordingly, four M. bovis CCs have been described (African 1 and 2, European 1 and 2), and these are determined based on specific deletions ranging from 806 to 14,094 bp (base pairs), SNPs and spoligotypes (Müller et al., 2009 Berg et al., 2011 Smith et al., 2011 Rodriguez-Campos et al., 2012). As with M. tuberculosis lineages, M. bovis CCs appear to have distinct geographical distributions, with African 1 and 2 restricted to Africa, European 2 commonly found in the Iberian Peninsula, and European 1 distributed globally (Müller et al., 2009 Berg et al., 2011 Smith et al., 2011 Rodriguez-Campos et al., 2012). Although there are no studies specifically aimed at identifying differences in virulence patterns among M. bovis of different CCs, numerous articles report virulence variations among strains of M. bovis (Wedlock et al., 1999 Waters et al., 2006 Meikle et al., 2011 Wright et al., 2013 de la Fuente et al., 2015 Vargas-Romero et al., 2016), suggesting a possible link between bacterial genetic polymorphisms and disease development, as observed in M. tuberculosis.

Since the whole-genome sequence of the first M. bovis strain became available in 2003 (Garnier et al., 2003), increasing efforts have been made to sequence additional strains and use whole-genome information to tackle bovine and/or wildlife TB transmission within specific outbreaks or countries (Bruning-Fann et al., 2017 Sandoval-Azuara et al., 2017 Ghebremariam et al., 2018 Kohl et al., 2018 Lasserre et al., 2018 Orloski et al., 2018 Price-Carter et al., 2018 Razo et al., 2018). However, no studies to date have comprehensively analyzed M. bovis genomes at a global scale to provide insights into its populational structure and evolution based on whole-genome information. Few studies that have compared transboundary M. bovis strains analyzed bacterial isolates obtained from a reduced number of countries (n < 9) and included small sample sizes (Dippenaar et al., 2017 Patané et al., 2017 Zimpel et al., 2017a Ghebremariam et al., 2018 Lasserre et al., 2018). Nevertheless, attained results suggest that M. bovis strains are likely to cluster based on geographical location (Dippenaar et al., 2017 Zimpel et al., 2017a Lasserre et al., 2018). In our previous study, we have also shown that few M. bovis genomes do not carry any CC genetic marker (Zimpel et al., 2017a), a phenomenon that was recently observed in M. bovis isolates from one cattle herd in the United States and from slaughterhouse cattle in Eritrea (Ghebremariam et al., 2018 Orloski et al., 2018). These findings suggest that CCs are unlikely to represent the whole diversity of M. bovis strains, warranting further evaluation of M. bovis molecular lineages (Zimpel et al., 2017a Lasserre et al., 2018). Therefore, the aims of this study were to perform a phylogenomic analysis to understand the populational structure of M. bovis worldwide and to provide dating estimates for the origin of this important pathogen.

We have screened over 2,600 publicly available M. bovis genomes and newly sequenced four wildlife M. bovis strains, gathering 1,969 M. bovis genomes from 23 countries and at least 24 different host species, including humans, to complete a phylogenomic analyses. Our phylogenetic reconstruction suggests the existence of at least four distinct lineages of M. bovis in the world. We also evaluated the evolutionary origin of M. bovis strains and lineages and correlated bacterial population dynamics with historical events to gain new insights into the widespread nature of bovine TB worldwide.


My research has centered on vertebrate population ecology at three levels: physiological processes, individual space use and resource selection, and population dynamics. I combine field studies with rigorous quantitative methods to address natural resource management issues. I want my research to advance science and our understanding of natural systems while impacting management and policy. Much of my current research and interest focuses on harvested wildlife populations of a broad range of taxa with a particular emphasis on large mammals. However, I routinely work on non-game species and my students have worked on species ranging from bees to hellbenders to elephants. I am driven by the research questions and relevance of work to policy and management. Our research is highly collaborative with state and federal agencies, non-governmental organizations, university faculty, and private industry. We have routinely received funding from a diversity of sources including many state and federal agencies, non-governmental organizations, National Science Foundation, and private industry.

2016 - North Central Section of The Wildlife Society Professional Award of Merit (presented for outstanding professional accomplishments in wildlife conservation)

2015 - E. Sydney Stephens Professional Lifetime Achievement Award (highest honor presented by Missouri Chapter of The Wildlife Society for outstanding contributions to wildlife management in Missouri)

2014 - Appointed Fellow of The Wildlife Society

2013 and 2014 - Superior Graduate Faculty Award, University of Missouri GraduateSchool and Graduate Student Organization

2013 and 2014 - Outstanding Fisheries and Wildlife Graduate Faculty, Department of Fisheries and Wildlife Sciences Graduate Student Organization, University of Missouri

2013 - Inaugural Recipient Southeastern Athletic Conference Faculty Achievement Award, University of Missouri (honors professors with outstanding records in scholarship and teaching who serve as role models for other faculty and students)

2009 - Missouri Governor&rsquos Award for Excellence in Teaching

2008 - U.S.D.A. National Teacher of the Year (U.S. Department of Agriculture, Excellence in College and University Teaching Award in the Agricultural Sciences &ndash National Level)

2007 - The Wildlife Society Award for Best Article (with Steve Buskirk)

2005 - Missouri Department of Conservation &ldquoOutstanding Research Collaborator of the Year&rdquo

2005 - William T. Kemper Fellowship for Excellence in Teaching, University of Missouri (most prestigious teaching honor at MU)


Results

The following chapter presents the insights gathered from the reconstruction of the historical dimension of wildlife trends and species occurrence gathered from the secondary data (literature and document) analysis followed by the expert interviews and community questionnaires.

Wildlife and human-wildlife conflict trends

Table 1 presents population estimates of the elephant, blue wildebeest, plains zebra, lion and African buffalo for all KAZA TFCA countries. Elephant populations in Namibia and the other KAZA countries have steadily increased since 1934. Plain’s zebra numbers considerably increased in Namibia, but decreased in Botswana, Zambia and Zimbabwe. While the Namibian lion and buffalo populations have remained at a constant level, lion populations in the other KAZA countries decreased. Buffalo numbers increased in Botswana and decreased strongly in Zimbabwe. After sharp declines in wildebeest populations around 1965, they are recovering in both Namibia and Botswana. A more detailed description of wildlife trends can be found in Supplementary Material 6.

Wildlife numbers in the table above represent species populations for the whole country. Figure 2 shows wildlife trends for four of the five studied species in the Namibian component of the KAZA TFCA. Unfortunately, no disaggregated data by region was available for the Plain’s Zebra and older surveys for the Zambezi and Kavango Regions do not exist. The data from 1995 to 2015/2016 shows that elephant numbers in the Namibian component of KAZA have more than tripled. The region hosts most of Namibia’s elephant population estimated at 22 754 in 2016. Lion populations have declined. However, this estimate for 2018 by the IUCN Cat Specialist Group was not based on new data. Both wildebeest and buffalo populations increased as well. No recent information or aerial surveys have been published since the study of Estes and East in 2009. However, wildebeest seem to recover after they reached their lowest level between the 1960s and 1980s 17 . Buffalo populations have also increased considerably since the 1960s. The estimate of the most recent buffalo population (5650 heads) is based on buffalo sightings during the 2018 conservancy game counts conducted by NACSO in the region. The buffalo population in 2018 is thus likely to be a severe underestimation as it does not account for buffalos living outside of conservancies and conservancies that have not been surveyed.

Wildlife trends for the elephant, blue wildebeest, plains zebra, lion and African buffalo in the Namibian component of the KAZA TFCA. The graph depicts the wildlife numbers for four species (elephant in blue, lion in green, wildebeest in yellow and buffalo in red ) in the three regions (Kavango East, Kavango West and Zambezi) that constitute the Namibian component of the KAZA TFCA.

Data on reported human–wildlife conflict incidents for all conservancies employing the Event Book System in Namibia and conservancies located in the KAZA TFCA over the last 15 years show different trends. Reported incidences of human–wildlife conflict in all investigated conservancies (Fig. 3) were initially low. From 2003 to 2013 the reported incidents increased and then decreased between 2013 and 2015. Reported incidents in conservancies within the KAZA TFCA (Fig. 3) increased until 2003. Thereafter, incidents remained relatively constant with upward and downward fluctuations. Taking into account all studied conservancies in Namibia, predation of livestock by wildlife is the main challenge. Considering data from the conservancies that lie within the KAZA TFCA alone, crop damage is reported more often (on average around 71% of all reported incidents between 2002 and 2015) than in the Namibian context. Crop damage has decreased in these conservancies since 2012 and livestock predation is increasing. Human attacks and other damages, for example to infrastructure, are much less common (less than 5% of all reported incidents).

Reported human-wildlife conflict incidents in all conservancies that lie within the KAZA TFCA (n = 21) and in all investigated Namibian conservancies (n = 85). Map (a) shows the trend in human-wildlife conflict in all communal conservancies in Namibia between 2001 and 2015, while map (b) shows the trend in incidents in the communal conservancies lying within the Namibian component of the KAZA TFCA. The graph shows incidents of livestock damage (yellow), crop damage (green), human attacks (black) and other incidents (dark blue) as well as the total incidents (blue).

Reconstruction of historical occurrence of selected species

Figure 4 shows the occurrence patterns of the five selected species in 1934, 1975 and the most recent range from the Atlas of Mammals 29 . Lion and elephant populations expanded their range between 1934 and 1975. Their most recent range is considerably smaller than in 1975 and they are increasingly restricted to protected areas in the Namibian component of the KAZA TFCA and around the Etosha National Park. In the past, the African buffalo had a limited range along the Okavango and the large rivers in the north-east. Today, there are no more buffalos along the Okavango River in north-central Namibia, but they occur all over the north-east. The range of the two migratory species, plain’s zebra and blue wildebeest, was considerably smaller in 1975 than 1934. However, both species were able to recover some of their former range due to reintroductions to farmland and private reserves.

Occurrence patterns of the elephant, lion, blue wildebeest, pains zebra and African buffalo in 1934, 1975 and the most recent range. The map shows the distribution of the elephant (a), lion (b), blue wildebeest (c), plains zebra (d) and African buffalo (e) in 1934 (purple), 1975 (blue) and the most recent range (pink) indicated in the Atlas of Mammals (Atlasing of Namibia Initiative, Environmental Information Service Namibia) mapped using QGIS 3.12 14 . The size of the points is a result of different grids used for the data collection, which is explained in more detail in the methodology and Supplementary Material 1. In addition, it shows the location of fences (red), rivers (blue) and protected areas (different shades of green with increasing protection).

Figure 4 shows larger fences in Namibia and Botswana. Furthermore, the wildlife dispersal areas for 1934, 1975 and the current range based on the Atlas of Mammals and Carnivores are mapped. Fences seem to have a considerable effect on elephant, buffalo and to a certain degree also blue wildebeest. Shortly after the construction of a game-proof fence, the so-called “veterinary cordon fence” or “Red Line in Namibia in 1975, the range of the blue wildebeest was severely restricted, apart from a few smaller populations that survived south of the fence. The Atlas of Mammals also suggests a considerable influence of fences on wildebeest range. The plain’s zebra range also seems to be considerably influenced by fences -especially in the north-east. For both zebra and wildebeest, outliers can be explained by reintroductions on private game farms. The effect of fences on the buffalo population seems to be significant. Buffalo range is cut off along the border fence with Botswana to the west, the Red Line to the south and the Caprivi fence in the Bwabwata National Park. In 1975, elephants occurred south of the Red Line. In the current range elephants are restricted to protected areas (Etosha NP, Khaudom NP, Mangetti NP). Within the Bwabwata National Park their range is cut off along the border with Botswana in the south-western part. Lion range is less influenced by the Red Line in the Etosha/Kunene area, where they occur on both sides of the fence, but is much more restricted in the north-east, where it is cut off by the border fence with Botswana, the Caprivi fence, as well as the Northern and Southern Buffalo fences in Botswana.

Human component: poverty, population density & land-use

Population density is high in the northern regions of Namibia compared to the rest of the country, especially in some of the former black “homelands” such as Ovamboland, Okavangoland and Eastern Zambezi (Fig. 5). Based on population and housing census data of the Namibia Statistics Agency, there has been an overall increase in population from around 1.4 million in 1991 to around 2.1 million in 2011. As a result, population density has increased across the country. For example, the population density in the Zambezi region has increased from 4.9 inhabitants/km 2 to 6.2 people/km 2 . Density in the Kavango region increased from 2.7 to 4.6 inhabitants/km 2 and from 0.9 to 1.4 inhabitants/km 2 in the Otjozondjupa region 30,31 .

Population density by region in 1991, 2001 and 2011 and the number of people classified as poor by region in 1995, 2003 and 2009. The figures show the population density (a) and poverty level (b) over time for northern Namibia mapped using QGIS 3.12 14 . Population density is indicated in people per square km: low values are indicated in dark blue and while high values are indicated in dark orange. Black homesteads -to which indigene communities were relocated under South African administration- are outlined in red. The level of poverty (% of people classified as poor) ranges from light green (low) to dark green (high). Light grey areas indicate national parks.

Based on the population censuses of the Namibia Statistics Agency (NSA), the number of people classified as “poor” has been reduced considerably since independence in 1990 (Fig. 5). The proportion of the population classified as poor was reduced in the Kavango (76.3% in 1993/4 to 55.2% in 2011) and Otjozondjupa regions (60.1% in 1993/4 to 33.7% in 2011). The Zambezi region, which had the highest proportion of “poor” people (81.3%) in 1991, also experienced a decrease in 2003/04 (36.5%). However, the proportion increased again to 50.2% in 2011 32 . Although poverty in the Namibian component of the KAZA TFCA has been reduced significantly since independence it remains at a relatively high level compared to the rest of the country.

Displaying data on irrigated areas, livestock density and cropping activity in Namibia shows considerable agricultural activity in the Namibian component of the KAZA TFCA (Fig. 1). High livestock densities (max. 10 livestock units/km 2 33 occur in the eastern Zambezi region, especially along the border with Zambia. Even within conservancies livestock densities can be high. In the central Zambezi region, a considerable area is under cultivation. Smaller areas with fields can be found all over the region and within conservancies. Two larger irrigated areas can be found in the Zambezi region, one in the Central Zambezi region and one within the Bwabwata National Park. Cattle are allowed within the multiple-use zones of the Bwabwata National Park and communities have small crop fields. Another “hotspot” of agricultural activity is along the border with Angola with relatively high livestock densities and a number of irrigated areas. The rest of the Namibian component has relatively low agricultural activity.

Expert interviews & community questionnaires

Expert Interviews were conducted with two representatives from the University of Namibia Department for Wildlife Management and Ecotourism, two local and two international NGOs, a government and a KAZA representative, as well as one external consultant and two representatives from community organisations. Two female experts and nine male experts were interviewed. Most participants, who answered the community survey, were between 25 and 35 years (46.3%) and 18 and 25 years (23.9%) old. Thus, the majority of respondents was relatively young and mostly male (64.2%). Ninety-five point one per cent of survey participants were members of a conservancy. The majority of respondents are members of the Kyaramacan Association in Bwabwata National Park (17), Dzoti (7) and Bamunu (7) conservancies. Surveys were also completed in Balyerwa, Kwandu, Mashi, Mayuni, Sobbe and Wuparo. Eleven respondents could not be assigned to a conservancy, either because they were not members or because they did not answer the question.

All experts agreed that the main challenge Namibia is facing is dealing with human–wildlife conflict. This increase was confirmed by communities living in the areas: Sixty per cent of the surveyed community members felt that human–wildlife conflict had increased over the past years, while 32.3% reported a decrease. The most common problems caused by wildlife were crop damage (87.9%) and livestock predation (69.7%). Loss of human life (18.2%) and damage to households (9.1%) were less frequent. The majority only experienced human–wildlife conflict a few times a year (59.1%). Others (18.2%) came into conflict a few times a month or every other month (12.1%). The elephant was stated as one of the main conflict species by 92.3% of respondents, followed by the buffalo (44.6%) and lions (43.1%). While the elephant was seen as a conflict species in every studied conservancy other species were more localised. For example, lions were reported as one of the main species causing major conflicts in Balyerwa, Bamunu, Dzoti, Sobbe and Wuparo, while inhabitants of the Bwabwata National Park, Mashi and Mayuni conservancy did not consider lions as a main conflict causing species in their area.

According to the experts, human–wildlife conflict can be driven by competition for food and space. Livestock is an important investment strategy for many local communities and livestock predation was thus a direct threat to their livelihood and survival. Wildlife, on the other hand, was a collective good. Not every community member benefited and in conjunction with poverty and hunger, this aggravated human–wildlife conflict. Communities, who indicated that they benefited very little, were mainly respondents who did not belong to a conservancy. Most respondents who indicated that they “strongly benefited” from wildlife also indicated that they “strongly valued” wildlife (p = 0.011, N = 61, Value = 21.346, df = 9). Respondents who indicated that they ‘benefit very little’ reported increases in human–wildlife conflict, while 56% of respondents who ‘strongly benefit’ perceived decreasing conflicts (p = 0.011, N = 61, Value = 16.675, df = 6).

All expert interview partners stated that wildlife numbers had increased considerably inside and outside of protected areas over the past years, although considerable numbers were poached. The increasing wildlife numbers reflect the success of the community based natural resource management (CBNRM) programme. This was confirmed by community members: When asked about ecological changes taking place in the region, 68.6% of survey respondents indicated that more wildlife was coming into the area in which they lived. This was especially the case for the Balyerwa, Bamunu, Dzoti, Sobbe and Wuparo conservancies. Groups of a particular species were increasing, according to 57.8% of respondents (17.8% no changes, 24.4% decrease) and species, that had not been in the area before were now coming to the area (56.1%).

According to experts, population density in the Namibian component of the KAZA region is high compared to the rest of the country and more people are moving into the area. Agricultural activities increase with the influx of people. Livestock is often mismanaged spurring further conflict, due to a lack of protective measures especially in an open system without fences. Generally, natural resources are becoming more important for the economy every year and wildlife, through tourism, has become one of the main contributors to the Namibian economy. Wildlife-based activities have a competitive advantage over other land-use activities, since they are better adapted to the (semi-) arid conditions. Community survey results suggest a significant association (p = 0.002, N = 37, Value = 17.146, df = 4) between changes in household income and changes in tourism. Cross-tabulation also confirms that income increased as tourism increased. The importance was likely to increase with the expected impact of climate change.

All interviewed experts were convinced that increasing wildlife and human populations are the main reason for an increase in human-wildlife conflict. However, most (55.1%) of surveyed community members associated increasing human–wildlife conflict with the presence of poachers followed by increasing wildlife populations (46.9%), increasing human populations (22.4%) and water scarcity (18.4%). Thirty of 34 survey respondents (88%) who claimed that poaching increased also reported increases in human–wildlife conflict. Eighteen of 27 (66%) respondents who claimed that poaching decreased reported decreases in human–wildlife conflict (p = 0.000, N = 65, Value = 31.018, df = 4).

According to the interviewed experts, once successfully implemented, the KAZA TFCA could improve the movement of wildlife. Furthermore, connectivity could positively support ecosystem functioning and enhance biodiversity. Communities agreed and believed that the KAZA TFCA would be “very successful” in conserving biodiversity (52.8%), conserving cultural heritage (41.5%), creating a network of interlinking protected areas (52.1%), becoming a prime tourism destination (51.2%) as well as reducing poverty (46.7%). Almost one-third (27.3%) of survey respondents appreciated the concept because it aims to create space for the free movement of wildlife and 20% hoped that KAZA would build capacity in natural resource management, to achieve a more sustainable coexistence of humans and wildlife. Experts suggest that by securing corridors wildlife from areas with very high wildlife densities could disperse into less crowded areas. This would reduce pressure on the land and natural resources and minimise interaction and conflict with people living in areas with high wildlife density. On the other hand, an open and connected system could encourage poaching. In addition, due to the increased movement of wildlife, Namibia could become a wildlife transit route and human–wildlife conflict could increase.

To successfully encourage the dispersal of wildlife across the member countries the conditions within the countries need to be the same. Common ground on veterinary diseases and fences have to be found. In some areas, fences protect wildlife from the invasion of cattle. Social problems would also have to be addressed more seriously to make the concept successful. In addition, ways to control illegal settlement and disease outbreaks have to be found as they spread faster over open borders. Bottom-up planning, awareness campaigns, capacity building and programmes targeting poverty and livelihood enhancement are required to change the attitude of people towards wildlife and the KAZA TFCA. Activities need to be communicated to local communities, so they could see the direct and indirect benefits, e.g. the construction of schools and clinics. This needs to go hand-in-hand with increasing benefits from tourism and hunting and employment opportunities. Local communities should be provided with alternatives to agriculture. Above all, for the KAZA TFCA to become successful, political stability in the region is required and joint strategies to control wildlife and forest crime have to be implemented. Poaching in the region was a transboundary issue and facilitated by open borders, different policies and legislation of the individual countries.


1. Mitochondrial DNA markers for wildlife conservation

Many of the conservation genetics studies have utilized the sequence information of mitochondrial DNA (mtDNA). The mitochondrial genome comprises a circular chromosome of DNA. Animal mtDNA ordinarily contains 36 or 37 genes two for rRNAs, 22 for tRNAs and 12 or 13 for subunits of multimeric proteins of the inner mitochondrial membrane. In addition, there is a noncoding sequence termed the control region (CR) due to its role in replication and transcription of mtDNA molecules. Exons in the mtDNA circle are tightly packed with no spacing introns. Mitochondrial DNA is histone-free, has limited repair ability, and therefore has a relatively high mutation fixation rate (5� times that of nuclear DNA). Although mtDNA has evolved faster than the nuclear genome, the rate of evolution is different for different regions of mtDNA and has been used to examine various phylogenetic relationships. Several conserved primers have been developed that allow amplification of a number of regions of the mtDNA molecule in a wide range of species. Moreover, because most cells contain multiple copies of the mtDNA molecule, mtDNA sequences can often be obtained from very small amounts of tissue containing degraded DNA. The main uses of mtDNA sequences in conservation genetics include population structuring, resolving taxonomies, establishing interspecific hybridization and the detection of illegal hunting and poaching of endangered animals. However, the selection of appropriate bioinformatics tool plays an important role for reliable phylogenic inference using mitochondrial markers (Khan et al., 2008b). Table 1 provides quick information on numbers of samples and populations, names of taxa and types of mitochondrial markers used in some relevant studies.

Table 1

Application of mtDNA markers for conservation of wild animals.

No. of samplesNo. of populationsTaxaStudyMarkerReference
211 CaptiveSun bearEvolutionary significant unitCROnuma et al. (2006)
473 PopulationsBlack muntjacPopulation structureCRWu et al. (2006)
401 PopulationChinese water deerGenetic diversityCRHu et al. (2006)
5Roe, deer, horse, cowIllegal huntingCyt bAn et al. (2007)
731 PopulationHoubara bustardGenetic diversityCRIdaghdour et al. (2004)
9517 LocationsAfrican sableGenetic structureCR, Cyt bPitra et al. (2002)
18 +ꁔ2 LocationsRock-wallabyPopulation structureCREldridge et al. (2001)
4610 SourcesTibetan gazelleGenetic diversityCR, Cyt bZhang and Jiang (2006)
17 +ꁂCaptive +ਏieldIndian leopardSpecies identification12S rRNAPandey et al. (2007)
18214 Zoos diff. countriesOryx dammahGenetic diversityCRIyengar et al. (2007)
19 +ꀖ +਄ +ਃ +ꀘ5 LocationsOryx beisaGenetic diversityCR, Cyt bMasembe et al. (2006)
21 +ਃ2 LocationsOryx leucoryxGenetic diversityCRKhan et al. (in press)

1.1. Ribosomal DNA (12S and 16S rDNA)

Mitochondrial 12s rDNA is highly conserved and has been applied to illustrate phylogeny of higher categorical levels such as in phyla or subphyla. Whereas the 16s rDNA is usually applied for phylogenetic studies at mid-categorical levels such as in families or genera. It has been postulated that these sequences are useful for inferring moderate to long divergence times (Janczewski et al., 1995). Several investigators have used 12S rDNA sequences for wildlife forensic biology (Prakash et al., 2000 Gurdeep et al., 2004). Molecular phylogeny of elopomorph fishes using 12S rRNA sequences clearly separated monophyletic Elopomorpha from Clupeomorpha (Wang et al., 2003). Shukla et al. (2001) amplified the 12S rRNA gene using universal primers and then cloned and sequenced the 450 bp fragment for phylogenetic analysis of endangered species, Indian muntjac (Muntiacus muntjak). The 12S rRNA gene has been used to examine genetic variation in the endangered spur-thighed tortoise (Testudo graeca), an endangered species from broad distribution range (Alvarez et al., 2000 van der Kuyl et al., 2005). The 12S fragment of Testudo graeca was found to be somewhat less variable than the D-loop fragment, a finding compatible with the situation in mammals, where rRNA genes evolve slower than synonymous sites and the variable parts of the D-loop (Pesole et al., 1999). Recently, Pandey et al. (2007) have utilized 12S rDNA for molecular identification of Indian leopard, which is an endangered species except in Central Africa and India.

Lei et al. (2003) have examined the mitochondrial rRNA genes of Chinese antelopes and observed that average sequence divergence values for 16S and 12S rRNA genes are 9.9% and 6.3% respectively. Their phylogenetic analysis has revealed that Przewalski’s gazelle is more closely related to Mongolian gazelle than Tibetan gazelle suggesting that the critically endangered Przewalski’s gazelle should be treated as a species, not a subspecies of the Tibetan gazelle, which clearly warrants more attention from conservationists (Lei et al., 2003). A single base in the 16S rDNA sequences from the endangered species Pinna nobilis has been found to be different in all analyzed individuals from a single population sample (Chios island) differentiating it from the others (Katsares et al., 2008). Mitochondrial 16S rRNA has been used to elucidate the pattern of relationships and systematic status of 4 genera, including 9 species of skates living in the Mediterranean and Black Seas (Turan, 2008). A heminested PCR assay based on species-specific polymorphism at the mitochondrial 16S rRNA gene has been designed for the identification of seven pecora species including Blackbuck, Goral, Nilgai, Hog deer, Chital, Sambar and Thamin deer (Guha and Kashyap, 2005). Molecular studies on endangered Pecoran have shown lower sequence diversity in 16S rRNA gene as compared to cytochrome b gene, both between and within species however the 16S rRNA gene harbored a larger number of species-specific mutation sites than cytochrome b gene, suggesting that it could be more useful for species identification (Guha et al., 2006). NaNakorn et al. (2006) have assessed the level of genetic diversity of critically endangered Mekong giant catfish species using sequences of 16S rRNA and detected 4 haplotypes among 16 samples from natural populations. These findings may have important implications for conservation of the Mekong giant catfish, especially in designing and implementing artificial breeding program for restocking purposes (NaNakorn et al., 2006). Recently, Khan et al. (2008a) have suggested the utility of 16S rRNA segment for molecular phylogeny of oryx at the genus and possibly species levels.

1.2. Mitochondrial protein coding genes

Compared to 12S and 16S rDNAs, the mitochondrial protein-coding genes evolve much faster and therefore regarded as powerful markers for inferring evolution history in lower categorical levels such as families, genera, and species. This feature of mtDNA in phylogeny is suitable for resolving taxonomic uncertainties in conservation genetics. Mitochondrial cytochrome b sequences have been used to understand the genetic diversity of Tibetan gazelle for better conservation planning (Zhang and Jiang, 2006). Partial mitochondrial cytochrome b genes of five mammalian specimens and Chromo-Helicase-DNA-binding (CHD) genes of five pheasants have been used to determine whether the specimens were from illegally hunted animals (An et al., 2007). Mitochondrial cytochrome b sequence analysis of cooked meat, remnants of the bird and the DNA obtained from the wooden chopping block revealed that the cooked meat and remnants of the bird were of a chicken, but the wooden chopping block was used to chop the meat of an endangered bird (Gupta et al., 2005). Partial cytochrome b based molecular phylogenetic trees and genetic distances have indicated that there is considerable genetic divergence between the Korean goral and the Chinese goral, but virtually none between Korean and Russian gorals suggesting the importance of molecular data for conservation of the goral populations of these regions (Min et al., 2004). Johnson and O𠆛rien (1997) have used NADH dehydrogenase subunit 5 together with 16S rRNA gene for phylogenetic analysis of multiple individuals of 35 species from Felidae family and recognized eight significant clusters or species clades that likely reflect separate monophyletic evolutionary radiations in the history of this family.

1.3. Non-coding or control region sequences

The control region (CR) is composed of three domains including ETAS (extended termination associated sequences) domain, central domain and CSB (conserved sequence block) domain. Each of the three domains presents a distinct pattern of variation both ETAS and CSB domains evolve rapidly whereas the central domain remains taxonomically conservative. Iyengar et al. (2006) have compared the CR sequences from several captive animals with the sequences for oryx species and the subsequent phylogenetic analysis of sequence variations revealed a close grouping of Oryx leucoryx with Oryx gazelle rather than Oryx dammah. Recently, Khan et al. (in press) have observed typical sequence variation in the CR gene of 23 captive-bred and reintroduced Oryx leucoryx samples in the form of 7 haplotypes one of these haplotypes has been reported earlier while the remaining 6 haplotypes are novel and represent different lineages from the founders. The CR sequences have been used to investigate the genetic status and evolutionary history of the Tibetan gazelle (Zhang and Jiang, 2006). Onuma et al. (2006) have sequenced the CR of the sun bear (Helarctos malayanus) using 21 DNA samples collected from confiscated animals to identify conservation units including evolutionarily significant units and management units. Wu et al. (2006) have used the partial CR (424 bp) sequences from 47 samples to assess the population structure and gene flow among the populations of black muntjac (Muntiacus crinifrons), a rare species endemic to China. The genetic diversity and population structure of the Chinese water deer have been investigated by analyzing the 403 bp fragment of the mitochondrial DNA CR revealing 18 different haplotypes in 40 samples (Hu et al., 2006). Idaghdour et al. (2004) have sequenced 854 bp of CR from 73 birds to describe their population genetic structure of Chlamydotis undulata, a declining cryptic desert bird whose range extends from North Africa to Central Asia. A single CR haplotype was identified in New Zealand population, while 17 haplotypes were found in Australian populations of brush-tailed rock-wallabies (Petrogale penicillata), which were introduced to New Zealand from Australia in the early 1870s and have experienced widespread population declines and extinctions (Eldridge et al., 2001).


Executive Summary

Why we need to publish a rule. Under section 4(d) of the Act, whenever any species is listed as a threatened species, we are required to issue any regulations deemed necessary and advisable to provide for the conservation of such species. Also, any species that is determined to be endangered or threatened under the Act requires critical habitat to be designated, to the maximum extent prudent and determinable. The Panama City crayfish is proposed as a threatened species under the Act, and this document proposes regulations we deem necessary and advisable under section 4(d) of the Act, and also proposes to designate critical habitat. Designations and revisions of critical habitat can only be completed by issuing a rule. In light of the time that has passed since the publication of the proposed listing rule and the receipt of new scientific information, we are also reopening the comment period for the proposed listing rule.

What this document does. We are concurrently reopening the comment period for the proposed listing rule, proposing a 4(d) rule, and proposing to designate critical habitat for the Panama City crayfish. A draft economic analysis on impacts expected from the critical habitat proposal is also available.

The basis for our action. Under the Act, we may determine that a species is an endangered or threatened species because of any of five factors: (A) The present or threatened destruction, modification, or curtailment of its Start Printed Page 19839 habitat or range (B) overutilization for commercial, recreational, scientific, or educational purposes (C) disease or predation (D) the inadequacy of existing regulatory mechanisms or (E) other natural or manmade factors affecting its continued existence. Our proposed rule identified habitat loss and fragmentation from development (Factor A) as a primary threat to the Panama City crayfish, making the species warranted for protection as a threatened species under the Act.

The Act provides a specific list of prohibitions for endangered species under section 9, but the Act does not automatically extend these same prohibitions to threatened species. Under section 4(d), the Act instructs the Secretary of the Interior (Secretary) to issue any protective regulations deemed necessary and advisable for the conservation of threatened species. It also indicates the Secretary may extend some or all of the prohibitions in section 9 to threatened species. We are proposing a 4(d) rule that specifically tailors measures that provide for the conservation of the Panama City crayfish.

Section 4(a)(3) of the Act requires the Secretary to designate critical habitat concurrent with listing to the maximum extent prudent and determinable. Section 3(5)(A) of the Act defines critical habitat as (i) the specific areas within the geographical area occupied by the species, at the time it is listed, on which are found those physical or biological features (I) essential to the conservation of the species and (II) which may require special management considerations or protections and (ii) specific areas outside the geographical area occupied by the species at the time it is listed, upon a determination by the Secretary that such areas are essential for the conservation of the species. Section 4(b)(2) of the Act states that the Secretary must make the designation on the basis of the best scientific data available and after taking into consideration the economic impact, the impact on national security, and any other relevant impacts of specifying any particular area as critical habitat.

We prepared an economic analysis of the proposed designation of critical habitat. In order to consider economic impacts, we prepared an analysis of the economic impacts of the proposed critical habitat designation. We hereby announce the availability of the draft economic analysis and seek public review and comment.

Peer review. In accordance with our joint policy on peer review published in the Federal Register on July 1, 1994 (59 FR 34270), and our August 22, 2016, memorandum updating and clarifying the role of peer review of listing actions under the Act, we sought the expert opinions of nine appropriate specialists regarding version 1.1 of the species status assessment (SSA) report, and four appropriate specialists regarding version 2.0 of the SSA report. We received responses from four specialists for each version (total of eight peer reviews), which informed this proposed rule. The purpose of peer review is to ensure that our listing determinations, critical habitat designations, and 4(d) rules are based on scientifically sound data, assumptions, and analyses. The peer reviewers have expertise in the species' biology, habitat, and response to threats.


Figure 1

Figure 1. Scale of potential effects in marine wildlife groups and forest biota based on data from ref 4 with indication of dose rates from exposure to 131 I, 134 Cs, and 137 Cs during the first month after the Fukushima accident.

The lack of a more severe impact to the forest ecosystem is partially due to the accident occurring in late winter, unlike the April 26 accident at Chernobyl. Had the Fukushima accident occurred in midspring, radiosensitive young of many species would have been exposed. The delay in reproduction and rapid decay of 131 I will result in bird eggs receiving some 20 mGy d –1 , rather than ∼70 mGy d –1 if the accident had occurred one month later. However, due to the uncertainties in these initial calculations, a survey of egg hatchability and survival of newborn mammals should be implemented. Moreover, the Fukushima forest zone will be a relevant observatory site for studying adaptation processes to chronic exposures that are 10 to 100× background, a key issue that has yet to be solved from the Chernobyl accident.(5)

In contrast to the forest ecosystem, our analyses indicate that more severe impacts are likely for the coastal ecosystem adjacent to the Fukushima nuclear plant. The marine calculations were performed in a similar manner, except that to include dose from external irradiation by contaminated marine sediments, radionuclide-specific distribution coefficients (Kdr from ERICA(3)) were used to estimate activity concentrations in marine sediments from the activity concentrations reported in seawater, where Kdr = (Cr sed )/(Cr wat ) (in L kg –1 ). Seawater concentrations of 131 I reached 180 000 Bq L –1 on March 30, with an associated 47 000 Bq L –1 of 137 Cs (measured 330 m offshore). Activity concentrations decreased rapidly with distance due to very high dilution in the seawater (ca. 1/1000, 30 km offshore), and with time: 137 Cs decreased by a factor 30 within two weeks and 131 I decreased by a factor of 200.

Nonetheless, maximum dose rates for 131 I, 134 Cs, and 137 Cs ranged from 210 to 4600 mGy d –1 the lowest for marine birds and the highest for macroalgae, with intermediate values of 2600 mGy d –1 for benthic biota—fish, molluscs, crustaceans. At such high dose rates, marked reproductive effects, and even mortality for the most radiosensitive taxa are predicted for all marine wildlife groups whose life history characteristics confine them to the near-field, contaminant release area. The high dose rates estimated by the models indicate that a field survey is urgently needed to verify radionuclide distributions within the coastal zone, to quantify the role of trophic transfers within food webs, and to determine the extent to which marine sediments will act as a secondary source of radionuclide uptake by biota.(1) All estimations were performed under the assumption of no additional marine releases after the end of March. Actual releases of unknown quantity appear to have continued past this date, thus our dose estimates may be low. Our estimates of dose rates are also under-predictions because they are based on measured data for only a few radioisotopes among the suite of possible radionuclides that composed the actual aquatic source terms (e.g., 58 Co, 95 Zr, 99 Mo, 99 m Tc, 105 Ru, 106 Ru, 129 m Te, 129 Te, 132 Te, 134 Cs, 136 Cs, 132 I, 140 Ba, 140 La).

For any postaccident ecological impact assessment of the Fukushima accident, great care will be needed in the quantification of radiation dose to biota, consideration of confounding effects (e.g., from the tsunami, complex mixture of toxicants), and careful sampling designs if meaningful results are to be obtained. The contaminated forests and marine ecosystems at Fukushima will be important long-term research sites for studying multigenerational effects from chronic exposures to low doses of radiation still a controversial topic—25 years after Chernobyl.(5)


Reconstruction of the 1918 Influenza Pandemic Virus

CDC researchers and their colleagues successfully reconstructed the influenza virus that caused the 1918-19 flu pandemic, which killed as many as 50 million people worldwide. A report of their work, &ldquoCharacterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus external icon ,&rdquo was published in the October 7, 2005 issue of Science. The work was a collaboration among scientists from CDC, Mount Sinai School of Medicine external icon , the Armed Forces Institute of Pathology, and Southeast Poultry Research Laboratory external icon . The following questions and answers describe this important research and related issues.

Note: For a detailed historical summary of this work, including how it was conducted, the people involved, and the lessons learned from it, see The Deadliest Flu: The Complete Story of the Discovery and Reconstruction of the 1918 Pandemic Virus.

Background on the Research

What research does the Science article describe? Why is it important?

Read more on how an expert group of researchers and virus hunters located the lost 1918 virus, sequenced its genome, and reconstructed the virus in a highly safe and regulated laboratory setting at CDC to study its secrets and better prepare for future pandemics.

This report describes the successful reconstruction of the influenza A(H1N1) virus responsible for the 1918 &ldquoSpanish flu&rdquo pandemic and provides new information about the properties that contributed to its exceptional virulence. This information is critical to evaluating the effectiveness of current and future public health interventions, which could be used in the event that a 1918-like virus reemerges. The knowledge from this work may also shed light on the pathogenesis of contemporary human influenza viruses with pandemic potential. The natural emergence of another pandemic virus is considered highly likely by many experts, and therefore insights into pathogenic mechanisms can and are contributing to the development of prophylactic and therapeutic interventions needed to prepare for future pandemic viruses.

What are the reasons for doing these experiments?

The influenza pandemic of 1918-19 killed an estimated 50 million people worldwide, many more than the subsequent pandemics of the 20th century. The biological properties that confer virulence to pandemic influenza viruses have not traditionally been well understood and warranted further study. Research to better understand how the individual genes of the1918 pandemic influenza virus contribute to the disease process provide important insights into the basis of virulence. This kind of information has helped health officials to devise appropriate strategies for early diagnosis, treatment, and prevention, should a similar pandemic virus emerge. Additionally, such research informs the development of general principles with which we can better design antiviral drugs and other interventions against all influenza viruses with enhanced virulence.

Who funded the work described in this article?

Work with the reconstructed 1918 virus was conducted at and supported by CDC. The U.S. Department of Agriculture (USDA), the National Institutes of Health (NIH), and the Armed Forces Institute of Pathology (AFIP) all provided support for many other aspects of this research.

When did CDC begin research on the 1918 virus?

CDC studies of the 1918 influenza virus were begun in 2004 with the initiation of testing of viruses containing subsets of the eight genes of the 1918 virus. Previous articles describing the properties of such viruses were published before 2005. Reconstruction of the entire 1918 virus was begun in August 2005.

Could a 1918-like H1N1 virus re-emerge and cause a pandemic again?

It is impossible to predict with certainty the emergence of a future pandemic, including a 1918-like virus. Pandemics occur when an influenza virus emerges to which there is little, or no, preexisting immunity in the human population. However, it is generally thought that a 1918-like pandemic would be less severe due to the advent of vaccines to prevent flu, current FDA-approved antiviral influenza drugs, and the existing global influenza surveillance system that the World Health Organization maintains.

Are current antivirals and vaccines effective against the 1918 H1N1 virus?

Yes. Oseltamivir (Tamiflu® or generic), has been shown to be effective against similar influenza A(H1N1) viruses and is expected to be effective against the 1918 H1N1 virus. Other antivirals (zanamivir, peramivir and baloxavir) have not been tested against this specific virus but are expected to also be effective. Vaccines containing the 1918 HA or other subtype H1 HA proteins were effective in protecting mice against the 1918 H1N1 virus. Vaccination with current seasonal influenza vaccines is expected to provide some protection in humans since seasonal influenza vaccines provided some level of protection against the 1918 H1N1 virus in mice.

Are new prophylactics and therapeutics that could be effective against the 1918 virus under way?

Scientists continue to work on development of new antivirals which may be effective against a 1918-like virus. The reconstruction of the 1918 H1N1 pandemic virus and subsequent studies that followed showed that the 1918 polymerase genes contribute to efficient replication of the pandemic virus. This insight identified an important virulence factor in the study of influenza that is now targeted for antiviral compound development. Therefore, new polymerase inhibitors promise to add to the clinical management options against influenza virus infections in the future.

Biosafety Precautions

Was the public at risk from the experiments being done on this virus?

The work described in this report was done using stringent biosafety and biosecurity precautions that are designed to protect workers and the public from possible exposure to this virus (for example, from accidental release of the virus into the environment). The 1918 virus used in these experiments has since been destroyed at CDC and does not pose any ongoing risk to the public.

What biosafety and biosecurity precautions for protecting laboratory workers and the public were in place while this work was being done?

Before the experiments were begun, two tiers of internal CDC approval were conducted: an Institutional Biosafety Committee review and an Animal Care and Use Committee review. All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled in accordance with biosafety guidelines of the Interim CDC-NIH Recommendation for Raising the Biosafety Level Laboratory Work Involving Noncontemporary Human Influenza Viruses. Although the 1918 virus was not designated as a select agent at the time this work was performed, all procedures were carried out using the heightened biosecurity elements mandated by CDC&rsquos Select Agent program. The Intra-governmental Select Agents and Toxins Technical Advisory Committee recommended that the reconstructed 1918 influenza virus be added to the list of HHS select agents on September 30, 2005. Following this recommendation, CDC amended its regulations and designated all reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 Influenza virus) as a select agent.

What are the appropriate biosafety practices and containment conditions for work with the 1918 strain of influenza?

Biosafety Level 3 or Animal Biosafety Level 3 practices, procedures and facilities, plus enhancements that include special procedures (discussed in the next question below), are recommended for work with the 1918 strain. There are four biosafety levels that correspond to the degree of risk posed by the research and involve graded levels of protection for personnel, the environment, and the community. Biosafety Level 4 provides the most stringent containment conditions, Biosafety Level 1 the least stringent. These biosafety levels consist of a combination of laboratory practices and techniques, safety equipment, and laboratory facilities that are appropriate for the operations being performed. The specific criteria for each biosafety level are detailed in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories.

What is Biosafety Level 3 &ldquoenhanced&rdquo? What are the specific enhancements used for work with the 1918 strain of influenza?

A Biosafety Level 3 facility with specific enhancements includes primary (safety cabinets, isolation chambers, gloves and gowns) and secondary (facility construction, HEPA filtration treatment of exhaust air) barriers to protect laboratory workers and the public from accidental exposure. The specific additional (&ldquoenhanced&rdquo) procedures used for work with the 1918 strain include:

  • Rigorous adherence to additional respiratory protection and clothing change protocols
  • Use of negative pressure, HEPA-filtered respirators or positive air-purifying respirators (PAPRs)
  • Use of HEPA filtration for treatment of exhaust air and
  • Amendment of personnel practices to include personal showers prior to exiting the laboratory.

Further details of the biosafety recommendations for work with various human and animal influenza viruses, including 1918 virus, can be found in the interim CDC/NIH guidance for such work at Interim CDC-NIH Recommendation for Raising the Biosafety Level Laboratory Work Involving Noncontemporary Human Influenza Viruses.

How were these experiments conducted safely using containment provided by BSL-3 with enhancements?

Highly trained laboratorians worked with the 1918 influenza virus strain safely using BSL-3-enhanced containment. Researchers at CDC receive specialized training and go through a rigorous biosafety (and security) clearance process. For the work reported in the Science article, the lead CDC researcher provided routine weekly written reports to CDC management officials, including the agency&rsquos Chief Science Officer, and was instructed to notify agency officials immediately of any concerns related to biosafety or biosecurity.

A BSL-3 facility with specific enhancements includes primary (safety cabinets, isolation cabinets, gloves, gowns) and secondary (facility construction) barriers to protect laboratory workers and the public from accidental exposure. Specific enhancements include change-of-clothing and shower-out requirements, and the use of a powered air purifying respirator (PAPR half body suits). The primary and secondary barriers plus additional personal safety practices provide appropriate containment for conducting such influenza research. CDC evaluated the specific studies to be conducted as well as the highly experienced scientific team conducting the research and concluded that this work could proceed under BSL-3 containment with enhancements.

Why was BSL-3-enhanced containment used for work on the 1918 H1N1 virus when most human influenza viruses of the H1N1 subtype are handled under much less stringent containment?

The appropriate biosafety measures for working a given pathogen depend upon a number of factors, including previous experience with the pathogen or similar pathogens, the virulence and transmissibility of the pathogen, the type of experiment, and the availability of vaccines and/or antimicrobial drugs effective against the pathogen. Prior to reconstruction of the 1918 virus, CDC carefully evaluated the specific studies to be conducted and concluded that this research could safely and securely be done under BSL-3-enhanced containment. All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled under high-containment (BSL 3-enhanced) laboratory conditions in accordance with guidelines of NIH and CDC. The recommendations for biosafety levels are made by a panel of experts and are followed in a stringent manner.

A higher level of containment (biosafety level 4) is utilized for work on novel or exotic pathogens for which there is no treatment or vaccine. This is not the case for the 1918 virus. Descendants of the 1918 influenza virus still circulate today, and current seasonal influenza vaccines provide some protection against the 1918 virus. In addition, two types of antiviral drugs, rimantadine (Flumadine) and oseltamivir (Tamiflu® or generic) have been shown to be effective against similar influenza A(H1N1) viruses and are expected to be effective against the 1918 H1N1 virus. Other antivirals (zanamivir, peramivir and baloxavir) have not been tested against this specific virus but also are expected to be effective.

The physical and engineering design of BSL-3-enhanced containment is very similar to that used in BSL-4 laboratories. The BSL-3 laboratory also has state-of-the-art directional airflow control which filters outgoing air, and all waste is autoclaved or decontaminated before it leaves the work area, preventing escape of infectious agents.

Biosecurity Issues

Did the generation of the 1918 Spanish influenza pandemic virus containing the complete coding sequence of the eight viral gene segments violate the Biological Weapons Convention?

No. Article I of the Biological Weapons Convention (BWC) specifically allows for microbiological research for prophylactic, protective, or other peaceful purposes. Article X of the BWC encourages the &ldquofullest possible exchange of&hellipscientific and technological information&rdquo for the use of biological agents for the prevention of disease and other peaceful purposes. Further, Article X of the BWC provides that the BWC should not hamper technological development in the field of peaceful bacteriological activities. Because the emergence of another pandemic virus is considered likely, if not inevitable, characterization of the 1918 virus may enable us to recognize the potential threat posed by new influenza virus strains, and it will shed light on the prophylactic and therapeutic countermeasures that will be needed to control pandemic viruses.

Did the report provide a &ldquoblueprint&rdquo for bioterrorists to develop and unleash a devastating pandemic on the world?

No. This report does not provide the blueprint for bioterrorist to develop a pandemic influenza strain. The reverse genetics system that was used to generate the 1918 virus is a widely used laboratory technique. While there are concerns that this approach could potentially be misused for purposes of bioterrorism, there are also clear and significant potential benefits of sharing this information with the scientific community: namely, facilitating the development of effective interventions, thereby strengthening public health and national security.

Is the 1918 influenza virus a select agent?

The Intra-governmental Select Agents and Toxins Technical Advisory Committee convened on September 30, 2005, and recommended that the reconstructed 1918 influenza virus be added to the list of HHS select agents. Following this recommendation, CDC amended its regulations and designated all reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 Influenza virus) as a select agent.

What is the Select Agent Program?

The Centers for Disease Control and Prevention (CDC) regulates the possession, use and transfer of select agents and toxins that have the potential to pose a severe threat to public health and safety. The CDC Select Agent Program oversees these activities and registers all laboratories and other entities in the United States of America that possess, use or transfer a select agent or toxin.


Watch the video: Reconstruction of Signals from Samples (January 2023).