1,721,096 research outputs found
Development of high-throughput metabarcoding approaches to explore genetic diversity and anthelmintic resistance in sheep gastrointestinal nematodes
Get PDFSheep farming in the UK, integral to its cultural and economic fabric, contends with
significant challenges from gastrointestinal nematodes (GINs) which impair livestock
productivity and increase mortality, leading to substantial economic losses. The
emergence of anthelmintic resistance, compounded by the impacts of climate change,
has necessitated integrated parasite management (IPM) strategies and the adoption of
advanced DNA technologies such as next-generation sequencing (NGS) for diagnosis
and targeted treatment.
In this project, rDNA ITS-2 and mt-ND4 genetic markers were employed to analyse GINs
across various locations, farms, and treatment groups. The ITS-2 analysis, also known
as the Nemabiome, provides robust identification and relative abundance of GIN species
when combined with faecal egg count, while mt-ND4 markers offer insights into the
origins, gene flow, and infection patterns of individual species. By leveraging these
technologies, this thesis aims to enhance the management and understanding of GINs,
contributing to the sustainability and productivity of UK sheep farming.
A significant portion of the work focused on refining these techniques. Despite the
effectiveness of Nemabiome sequencing in detecting all clade V nematodes, identifying
lesser-known GIN species remains challenging due to reference sequence gaps,
particularly in environments with cross-infections from GINs infecting diverse grazing
animals. To address this, the reference library was expanded using various GIN sample
sets, listing anticipated GIN species, extracting their ITS-2 rDNA sequences from NCBI
GenBank, and enhancing the library through iterative BLAST searches and phylogenetic
analyses.
Concurrently, mt-ND-4 markers were developed for T. circumcincta and H. contortus, the
two most prevalent GIN species in the UK. These mitochondrial markers, typically
species-specific, were adapted into a primer multiplex to process both species
simultaneously, significantly increasing efficiency and reducing costs. Reference libraries
were developed for both species, along with the post-sequencing analysis pipelines, and
the multiplex was tested on various laboratory strains, verifying its accuracy and reliability.
To implement both these techniques in the field, a DNA biobank was established,
collecting over 250 field samples from across Scotland and England to enable
comprehensive comparisons across various locations, treatments, and farms. This
biobank was further enhanced by the inclusion of nine characterised laboratory strains of
H. contortus and seven of T. circumcincta to assist in the comparison of resistance
mechanisms and genetic diversity.
Nemabiome analysis identified seven GIN species within the UK, with T. circumcincta
being the most prevalent, followed by H. contortus and Trichostrongylus vitrinus.
Together, these three species accounted for approximately 88% of infections. Significant
spatial differences in GIN distribution were observed between areas, with T. circumcincta
being prevalent in Scotland and H. contortus in England, likely influenced by climatic
conditions. Despite its generally lower presence, H. contortus was consistently found on
some Scottish farms, suggesting an influence of local environmental or management
factors. It also showed a higher prevalence and diversity in alpacas compared to sheep
or other hosts, raising questions about the role of alpacas as a maintenance host.
The effectiveness of levamisole, ivermectin, and their combined anthelmintic treatments
was assessed through faecal egg count reduction tests (FECRT) across 18 farms in
England. Treatments were more effective against some species than others: Resistance
was indicated in T. circumcincta, while Trichostrongylus axei and Trichostrongylus
colubriformis displayed mixed results. Notably, the combination treatment proved less
effective than individual drugs on some farms, which could suggest antagonistic
interactions between these drugs.
In the mt-ND4 analysis, 60 H. contortus and 35 T. circumcincta amplicon sequence
variants (ASVs) were identified, with a single ASV representing over one-third and over
62% of the total reads, respectively. Phylogenetic analysis suggested a common origin
for H. contortus, but potentially two distinct origins for T. circumcincta. H. contortus
showed a general trend towards balancing selection or population contraction across both
areas, while T. circumcincta showed population expansion or purifying selection in
Scotland and balancing selection or contraction in England. There was significant gene
flow with minimal genetic differentiation between Scottish and English populations across
both species.
Post-treatment analysis revealed a reduction in genetic diversity for H. contortus but the
opposite for T. circumcincta. Ivermectin-treated groups displayed higher H. contortus
diversity compared to those treated with levamisole, with the combination yielding
moderate diversity levels, supporting the idea of possible antagonistic interactions. No
difference was observed between treatment groups for T. circumcincta.
Overall, the study underscores regional and inter-species variations, alongside the
challenges of emerging anthelmintic resistance, especially in T. circumcincta. It highlights
the critical need for continuous monitoring and targeted management strategies to
effectively control GIN prevalence and manage resistance within UK sheep farming.
Additionally, the findings lay a foundation for utilising more diverse and specific genetic
markers to further investigate genetic diversity and resistance mechanisms
Study of gastrointestinal nematodes co-infecting feral Soay sheep on St Kilda
Get PDFThe unmanaged, feral Soay sheep population on St Kilda has survived for hundreds of years,
despite enduring potentially deleterious gastrointestinal nematode co-infections. Co-infections
with multiple nematode species are ubiquitous in feral and managed ruminants. Within these
mixed burdens, the different species may vary in their pathogenicity, epidemiology and clinical
presentation. Elucidating the diversity of different parasite species in a host, rather than
studying them as a homogenous group, is a prerequisite to understanding host-parasite
interactions. The primary aim of this thesis is to explore and validate non-invasive conventional
and next generation molecular parasitological methods to identify and quantify mixed-species
infections in feral hosts.
Seasonal patterns of gastrointestinal nematode parasitism in the Soay sheep were investigated
by faecal egg counts (FEC). Two FEC datasets were analysed: a large dataset, collected between
1988 and 2014, and counted by a modified McMaster method where each egg count represents
100 eggs per gram (epg); and a smaller dataset collected over one year between April 2015
and April 2016, counted by cuvette salt floatation method with a lower detection limit of 1 epg.
FEC generally declined with increasing sheep age, until the animals became geriatric (8 years +).
Seasonal FEC patterns in females generally followed a decline over the year starting in Spring.
FEC were generally higher in males, regardless of their age category, with little seasonal
variation between summer and winter once they became adults (3 years +). Monte Carlo
simulations were run in order to compare the effects of different detection limits between the
FEC methods detecting eggs in faeces to 100 epg and to 1 epg. The simulations suggest that the
method with the detection limit of 50 – 100 epg over-estimates the true egg counts within the
samples (at high counts), resulting in data that is highly negatively skewed and with an inflated
mean. Despite within-year variation of egg counts, both datasets resulted in overall similar
seasonal and host patterns. A shorter study with fewer replicates may benefit from a FEC
method with a lower detection threshold.
The eggs of most strongyle nematodes are morphologically similar, hence whilst FEC can
identify general trends in parasitology, the method provides limited information about the
proportions of mixed gastrointestinal nematode species burdens of the Soay sheep. The
development of advanced molecular methods for the in-direct genus or species-specific
diagnosis of strongyle infections in ruminants has negated many of the issues arising from
traditional parasitological techniques. Chapter 3 compared two molecular methods; a semi
automated multiplex-tandem PCR (AusDiagnostics™) with ITS-2 rDNA next-generation amplicon sequencing (nemabiome assay), to identify species (presence/absence), and quantify the
relative proportion (%) of ovine strongyle species in naturally infected samples. This chapter
provides the first, and preliminary, comparison of the sensitivity and quantitative ability of both
methods. There was good agreement between both molecular tests in determining the
presence/absence of species within a sample, but the correlation in their ability to quantify
relative proportions of the species present was moderate (C. ovina, R2 = 0.6096) to poor (Trichostrongylus spp., R2 = 0.2334). AusDiagnostics™ characterises a priori strongyle species
present, which partially suits a diagnostic tool for managed ruminant systems. The nemabiome
assay proved to be the most effective method for identifying and quantifying previously
unknown strongylid species, such as those associated with a feral host.
The sensitivity, bias, and repeatability of the nemabiome assay was tested for the strongylid
species identified in the Soay sheep of St Kilda (Chapter 4). A correction factor was calculated
for each species in order to reduce potential species-specific sequencing bias. This was
subsequently applied to the field data presented in Chapter 5.
Chapter 5 provides an epidemiological survey of the strongyle nematode species co-infecting
the Soay sheep of St Kilda. Teladorsagia circumcincta, Trichostrongylus axei, Trichostrongylus
vitrinus, Chabertia ovina and Bunostomum trigonocephalum were identified by the nemabiome
assay. The study highlights epidemiological trends in the Soay sheep that had not previously
been identified using conventional gross and molecular parasitological methods. There were
seasonal, age and sex differences in species proportions; whereby trends appeared to
correspond with the dynamic life-history of the sheep. Trends included a year round prevalence
of all species in lambs (males and females) to the age of one year old, high levels of T.
circumcincta over summer and winter in females older than 8 years, and no clear seasonality to
T. axei which persisted, at low levels, over all months in all sex/age groups sampled. However,
when relative species proportions were adapted to account for average FEC, the impact of the
trends seen was minimised; the adequate representation for individual nematode species relied
on a high faecal egg count. This is the first study to use the nemabiome deep amplicon
sequencing approach to characterise seasonal patterns in different co-infecting gastrointestinal
nematodes in feral sheep. Additionally, it highlights the flexibility of the nemabiome assay as a
viable non-invasive tool for parasitological surveys of wild animals.
Better knowledge of the epidemiology of different co-infecting gastrointestinal nematodes in
the ancient feral St Kilda Soay sheep population could help to explain the impact of grazing
management and anthelmintic drug treatments in managed sheep flocks, aiding in the
development of sustainable control strategies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Investigation of genetic and non-genetic factors influencing ewe reproductive performance in a Scottish hill flock
Get PDFHill sheep farming is the backbone of the Scottish sheep farming industry. Due to the low productivity of hill sheep breeds and the changes in EU subsidy support system, the number of breeding ewes declined significantly between 2000 and 2018. This has affected the social, economic and environmental sustainability of rural areas. Introduction of lowland/upland prolific sheep breeds, such as the Lleyn, into hill farming systems might improve productivity of hill enterprises. However, the harsh hill weather conditions and poor nutritional supply in the hill environment might compromise the performance and the health of less adapted breeds, such as the Lleyn. The aim of this PhD project was to investigate the performance of Lleyn ewes farmed in a Scottish hill farm environment, comparing their performance with genetically unimproved Scottish Blackface (UBF) ewes and genetically improved Scottish Blackface (IBF) ewes farmed together in the same flock.
The study was conducted between November 2012 and October 2017 in two phases. In the first phase (between November 2012 and October 2015; the flock was comprised of approximately 300 ewes per genetic line), Lleyn ewes achieved significantly higher litter sizes at pregnancy scanning, lambing and weaning than UBF and IBF ewes, and had heavier average lamb birth weight than, and comparable average lamb weaning weight to, UBF and IBF ewes. In the second phase (between November 2015 and October 2017; the flock was comprised of approximately 200 ewes per genetic line), the three genetic lines of ewes were further challenged, with half of the flock being farmed under more extensive conditions. In this phase, based on the whole flock performance, Lleyn ewes achieved comparable litter sizes at pregnancy scanning, lambing and weaning to UBF and IBF ewes. They also achieved heavier average lamb birth weights and heavier weaned litter weights than UBF and IBF ewes.
Further investigations were performed to discover the possible reasons that led to performance differences among the three genetic lines. Firstly, Lleyn ewes were found to have higher pre-mating serum concentrations of vitamin D (25(OH)D3 in particular) than UBF and IBF ewes, and these vitamin D parameters were positively associated with lamb birth weight in the following lambing season. Additionally, pre-lambing metabolic profiles of twin-bearing ewes showed that they were well nourished in late pregnancy, with Lleyn twin-bearing ewes having higher magnesium concentrations than UBF and IBF ewes, which suggested that these Lleyns had higher feed intake. These outcomes could potentially assist Lleyn ewes to give birth to lambs with relatively heavier birth weight, which enhances lamb survival. The quality of colostrum secreted by Lleyn twin-bearing ewes, measured as Brix percentage, was as good as colostrum secreted by UBF and IBF twin-bearing ewes, and this is important for providing passive immunity and energy for neonates to survive. The post mortem examinations of the majority of dead lambs in three lambing seasons (2015-2017) showed that dystocia was the main cause of neonatal lamb death, with more IBF lambs dying as a consequence of this cause, compared to UBF and Lleyn lambs. Lleyn ewes had narrower external pelvic widths than UBF and IBF ewes, although no relationship between this measurement and lambing difficulty was found. Summer grazing behavioural observations showed that the number of ewes observed in different grazing sectors did not differ among the three genetic lines, suggesting no obvious differences in grazing behaviour. Overall, this thesis showed that Lleyn ewes have adapted well to the hill environment, and outperformed/equalled production levels of their UBF and IBF flockmates. Therefore, this lowland/upland sheep breed could be a good candidate for improving the productivity of hill sheep farming enterprises
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Fasciola hepatica infection in sheep: current and novel diagnostic tests
Get PDFFasciola hepatica infections cause morbidity and mortality in sheep and have a
significant economic impact on farmers. The commonly used diagnostic tests; faecal
egg count (FEC), anti-Fasciola antibody ELISA (AbELISA) and the biochemical
assays (measuring GLDH and GGT) all have limitations, particularly in detection of
pre-patent infections in sheep. A coproantigen ELISA (cELISA) is reported to detect
low burdens of infection from 4 weeks post-challenge (wpc) and to only detect
current infection. A faecal PCR has been used for early detection of infection, but is
limited by inhibitory factors in faecal samples. Loop-mediated isothermal
amplification (LAMP) is more resistant to inhibitory factors and has the potential to
be a pen-side assay. Triclabendazole (TCBZ) is the drug of choice to treat immature
F. hepatica but there have been increasing reports of TCBZ treatment failure in the
UK. Treatment outcome is determined using a FEC reduction test (FECRT). A
cELISA reduction test (CRT) has recently been proposed.
Within this thesis the cELISA, along with FEC, and where feasible the AbELISA
and the use of GLDH and GGT concentrations, are evaluated in (1) an experimental
challenge model in sheep, (2) individual naturally exposed sheep, in early infection,
pre- and post-treatment situations, (3) groups of naturally exposed sheep, including
composite samples, in pre- and post-treatment situations and evaluating the FECRT
and CRT, lastly a LAMP assay is developed for the detection of F. hepatica, and
evaluated against cELISA, FEC and PCR based detection.
Two groups of 6 sheep were challenged with F. hepatica metacercarial cysts. In
both studies, AbELISA was first to detect infection (3-4 weeks post-challenge
(wpc)), followed by cELISA (3-10 wpc) and then FEC (9-10 wpc). Minor
fluctuations were seen in both FEC and cELISA levels over both studies and a
transient increase in cELISA levels was seen in the first study at 3-8 wpc. All
animals were dosed with TCBZ 2 weeks prior to slaughter. The highest FECR was
37% and all sheep had live fluke present in their livers post-mortem.
27 lambs were sampled monthly between June and November with AbELISA,
GLDH, GGT, FEC and cELISA tests performed. GLDH and GGT concentrations
were above reference ranges from June. AbELISA detected infection in most animals
by September and in all but one animal by November. FEC and cELISA both
detected some very early positive results, most likely false-positive results, but the
majority of animals became positive in November. Twelve lambs were followed to
slaughter and all had low burdens of fluke (≤10).
A cross-sectional study was conducted including 36 British farms, comprising 812
and 528 sheep pre- and post-treatment, respectively. Low FEC and cELISA results
were seen, with better agreement between the two tests pre- than post-treatment.
Disagreements between the two tests were more frequently seen where the FEC
detected infection but the cELISA did not. This was true both before and after
treatment.
80 animals from 2 Scottish farms were confirmed to be infected with liver fluke and
given either a TCBZ or closantel treatment and followed for 56 days. A closantel
treatment was given to animals that were still infected at 21 days post-treatment
(dpt). The highest FECR and CR of the TCBZ-treated groups was 60.3% and 56.4%,
respectively, and the lowest FECR and CR of the closantel-treated groups was 83.7%
and 94.9%, respectively. A small proportion of closantel-treated animals maintained
a low FEC following treatment. Both the FECRT and CRT indicated treatment
outcome from 7 dpt.
In a postal survey, 41 sample packs were sent to British farmers, of which 25 farmers
participated. Samples from 44 and 36 groups were submitted pre- and post-treatment,
respectively. Individual and composite faecal samples from each group were tested
by FEC and cELISA. Group mean FECs were low and prevalence of infection on
farms did not follow a normal distribution. The composite cELISA was more
sensitive than the average cELISA, whilst the opposite was true for FEC. The
composite cELISA was less sensitive than the composite FEC in low burden
situations. A modified version of the composite CRT showed good agreement with
the composite FECRT and appears promising in situations where burden was
sufficiently high.
A faecal LAMP assay, specific to F. hepatica, was developed and evaluated using
samples from one of the groups of 6 experimentally challenged animals described
above. FEC, cELISA and PCR testing were also performed and compared to the
LAMP results. LAMP first detected infection at 3 wpc, followed by cELISA (7 wpc),
FEC (10 wpc) and PCR (13 and 14 wpc).
The studies within this thesis (1) confirm that cELISA can detect experimental
infection of sheep with F. hepatica later than AbELISA but earlier than FEC, and
confirm the TCBZ resistant status of a British isolate (Moredun isolate), (2)
demonstrate that in animals naturally exposed to F. hepatica, the cELISA does not
have an advantage of earlier detection over FEC and is not as sensitive as FEC in
established infections (3) show that the modified CRT and composite CRT appear to
give a good indication of treatment outcome from 7 dpt, but is of limited use in
flocks with a low burden of infection, and (4) demonstrate that a faecal LAMP can
detect F. hepatica infection at 3 wpc
Improved use of abattoir information to aid the management of liver fluke in cattle
Get PDFFasciolosis, caused by the trematode parasite Fasciola hepatica, is a multi-host parasitic
disease affecting many countries worldwide. It is a well-recognized clinically and
economically important disease of food producing animals such as cattle and sheep. In
the UK, the incidence and distribution of fasciolosis has been increasing in the last decade
while the timing of acute disease is becoming more variable and the season suitable
for parasite development outside the mammalian host has been extended. Meanwhile
control is proving increasingly difficult due to changing weather conditions, increased
animal movements and developing anthelmintic resistance.
Forecasting models have been around for a long time to aid health planning related to
fasciolosis control, but studies identifying management related risk factors are limited.
Moreover, the lack of information on the accuracy of meat inspection and available
liver fluke diagnostic tests hinders effective monitoring of disease prevalence and treatment.
So far, the evaluation of tests available for the diagnosis of the infection in cattle
has mainly been carried out using gold standard approaches or under experimental settings,
the limitations of which are well known. In cattle, the infection mainly manifests
as a sub-clinical disease, resulting in indirect production losses, which are difficult to
estimate. The lack of obvious clinical signs results in these losses commonly being
attributed to other causes such as poor weather conditions or bad quality forage. This
further undermines establishment of appropriate control strategies, as it is difficult to
convince farmers to treat without demonstrating clear economic losses of sub-clinical
disease.
This project explores the value of slaughterhouse data in understanding the changing
epidemiology of fasciolosis, identifying sustainable control measures and estimating
the effect of infection on production parameters using data collected at one of the
largest cattle and sheep abattoirs in Scotland. Data used in this study include; a) abattoir
data routinely collected during 2013 and 2014, b) data collected during 3 periods
of abattoir based sampling, c) data collected through administration of a management
questionnaire and d) climatic and environmental data from various online
sources.
A Bayesian extension of the Hui Walter no gold standard model was used to estimate
the diagnostic sensitivity and specificity of five diagnostic tests for fasciolosis in cattle,
which were applied on 619 samples collected from the abattoir during three sampling
periods; summer 2013, winter 2014 and autumn 2014. The results provided novel
information on the performance of these tests in a naturally infected cattle population
at different times of the year. Meat inspection was estimated to have a sensitivity of
0.68 (95% BCI 0.61-0.75) and a specificity of 0.88 (95% BCI 0.85-0.91). Accurate
estimates of sensitivity and specificity will allow for routine abattoir liver inspection to
be used as a tool for monitoring the epidemiology of F. hepatica as well as evaluating
herd health planning.
Linear regression modelling was used to estimate the delay in reaching slaughter weight
in beef cattle infected with F. hepatica, accounting for other important factors such as
weight, age, sex, breed and farm as a random effect. The model estimated that cattle
classified as having fluke based on routine liver inspection had on average 10 (95% CI
9-12) days greater slaughter age, assuming an average carcass weight of 345 kg. Furthermore,
estimates from a second model indicated that the increase in age at slaughter
was more severe for higher fibrosis scores. More precisely, the increase in slaughter
age was 34 (95% CI 11-57) days for fibrosis score of 1, 93 (95% CI 57-128) days
for fibrosis score 2 and 78 (95% CI 30-125) days for fibrosis score 3. Similarly, in a
third model comparing different burden categories with animals with no fluke burden,
there was a 31 (95% CI 7-56) days increase in slaughter age for animals with 1 to 10
parasites and 77 (95% CI 32-124) days increase in animals with more than 10 parasites
found in their livers.
Lastly, a multi-variable mixed effects logistic regression model was built to estimate
the association between climate, environmental, management and animal specific
factors and the risk of an animal being infected by F. hepatica. Multiple imputation
methodology was employed to deal with missing data arising from skipped questions
in the questionnaire. Results of the regression model confirmed the importance of temperature,
rainfall and cattle movements in increasing the risk for fasciolosis, while it
indicated that the presence of deer can increase the risk of infection and that male cattle
have a reduced risk of infection.
Overall, this project has used slaughterhouse data to fill important knowledge gaps regarding
F. hepatica infection in cattle. It has provided valuable information on the accuracy
of routine abattoir meat inspection, as well as other diagnostic tests. It has also
provided estimates of the effect of infection on the time cattle take to reach slaughter
weight at different levels of infection and identified relevant risk factors related to the
infection. In conclusion, knowledge of the effect of infection on slaughter age, as well
as regional risk factors for F. hepatica infection, along with an improved use of abattoir
inspection results in the evaluation of treatment strategies, can provide farmers and
veterinarians with better incentives and tools to improve their herd health strategies and
in the longer term help reduce the incidence of liver fluke in cattle
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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