TETRACYCLINE LABELED BONE CONTENT ANALYSIS OF ANCIENT NUBIAN
REMAINS FROM KULUBNARTI
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Arts in the
Graduate School of The Ohio State University
By
Julie Anna Margolis
Graduate Program in Anthropology
The Ohio State University
2015
Master's Examination Committee:
Dr. Clark Spencer Larsen, co-advisor
Dr. Samuel D. Stout, co-advisor
Dr. Douglas E. Crews
Copyrighted by
Julie Anna Margolis
2015
ABSTRACT
Armelagos and colleagues (2001) have hypothesized that beer is a conduit for in
vivo tetracycline consumption by ancient Nubians. Streptomycetes bacteria has a high
prevalence in Sudanese-Nubian soil (60 -70%) and secretes the antibiotic under harsh
conditions such as fermentation. At the site of Kulubnarti, 21-S-46 cemetery (716 CE)
skeletons likely represent a working underclass contemporaneous with the 21-R-2
cemetery (752 CE) containing the remains of a land-owning class. Interpretations of
archaeological and osteological evidence suggest that poorer health and higher mortality
occurred in the S population. To test whether an anticipated difference in tetracycline
ingestion between S and R cemetery populations existed, the amount of tetracyclinelabeled bone was quantified under ultra violet light using image analysis software.
Amount of tetracycline labeling was expressed in terms of the total area of labeled bone
tissue in square micrometers, number of labeled osteons, and number of grid intersections
over labeled bone. No significant differences in percent tetracycline-labeled bone tissue,
or percent labeled osteons was observed between cemeteries. These results suggest that
tetracycline ingestion was similar for S and R group members, class differences were not
mediating tetracycline ingestion, and both sub-groups had equal access to beer.
ii
Dedicated to my mentor, George J. Armelagos.
iii
ACKNOWLEDGMENTS
This thesis would not have been possible without support from my parents,
friends, and mentors. I would like to thank my committee co-chairs Dr. Samuel D. Stout
and Dr. Clark S. Larsen, committee member Dr. Douglas E. Crews, and Dr. Mark Hubbe
for their guidance and helpful comments regarding my writing and presentation of
statistics. I am grateful to my friend Sara Becker (MSc. Maastricht University) for
translating Pipenbrink’s 1983 work from German to English for me. I extend thanks to
the Department of Anthropology at Emory University for allowing me to use their
facilities and resources for data collection. I thank Nicole L. Henderson for having been
an incredible lab assistant and for helping me test my proposed technique of image
analysis by re-measuring several samples as part of a preliminary inter-observer study.
Most of all, I would like to thank Dr. George J. Armelagos and Dr. Dennis P. Van
Gerven, as without them this project would not have been possible. I am extremely
grateful to Dennis for providing the Kulubnarti rib cross-sections for me to work with in
the first place, helping me work through and understand my statistics, and answering a
vast array of questions on his previous studies of ancient Nubia. As for George, I cannot
express how thankful and appreciative I am for everything he did for me. George gave
me this project to work on originally, gave me access to all of his equipment and
resources (including funding for materials), answered my endless questions, acted as a
iv
sounding board for me to develop further research questions, and provided guidance as
only a friend and mentor can in their ideal form. George prepared me for graduate school
and a career in anthropology, and without him I would not be where I am today. I am also
thankful that, while George did not see the final written version of this thesis before his
death (only earlier drafts), he was able to see this project of ours through to the end of my
analysis and the future directions of my research. I will be forever grateful to George
Armelagos, for everything.
v
VITA
2008................................................................Diploma, Libertyville High School
2010................................................................A.A. Anthropology, Oxford College of
Emory University
2012................................................................B.A. Anthropology, Emory University
2012 to present ..............................................Graduate Student, Department of
Anthropology, The Ohio State University
Publications
Margolis JA, Van Gerven DP, and Armelagos GJ. 2013. Tetracycline labeling in early
Christian burials from Kulubnarti, Nubia: Measure of class differences. American Journal
of Physical Anthropology Supplement 56, 2013 (Annual Meeting Issue):189.
Fields of Study
Major Field: Anthropology
vi
Table of Contents
ABSTRACT ........................................................................................................................ ii
ACKNOWLEDGMENTS ................................................................................................. iv
VITA .................................................................................................................................. vi
Publications ........................................................................................................................ vi
Fields of Study ................................................................................................................... vi
Table of Contents .............................................................................................................. vii
List of Tables ..................................................................................................................... ix
List of Figures ..................................................................................................................... x
CHAPTER 1: INTRODUCTION ...................................................................................... 1
Purpose ............................................................................................................................ 1
Background of Kulubnarti............................................................................................... 2
Health of the Kulubnarti Populace .................................................................................. 5
Nubian Antibiotics .......................................................................................................... 7
Why Study Tetracycline Consumption at Kulubnarti? ................................................. 10
CHAPTER 2: MATERIALS AND METHODS .............................................................. 12
vii
Materials ........................................................................................................................ 12
Methods ......................................................................................................................... 14
New Technique of Image Analysis Method .............................................................. 14
Osteon Count Method ................................................................................................ 17
Point-Count Method .................................................................................................. 17
CHAPTER 3: RESULTS .................................................................................................. 19
CHAPTER 4: DISCUSSION ............................................................................................ 24
CHAPTER 5: CONCLUSIONS ....................................................................................... 31
REFERENCES ................................................................................................................. 33
APPENDIX A: RAW DATA ........................................................................................... 37
APPENDIX B: RIB CROSS-SECTION PHOTOS .......................................................... 39
viii
List of Tables
Table 1: Sample specimens and associated sex and age estimations from Kulubnarti .... 13
Table 2: Percent averages of tetracycline-labeled bone for all ages at Kulubnarti ........... 20
Table 3: Percent averages of tetracycline-labeled bone among those aged 12 and older at
Kulubnarti ......................................................................................................................... 23
Table 4: Kulubnarti cemetery 21-S-46 (S Cemetery) raw data ........................................ 37
Table 5: Kulubnarti cemetery 21-R-2 (R Cemetery) raw data…………………..………38
ix
List of Figures
Figure 1: Map of Nubia (Turner et al. 2007) ...................................................................... 3
Figure 2: Tetracycline molecule ......................................................................................... 8
Figure 3: Rib cross-section from specimen S48 before digital cleaning .......................... 15
Figure 4: Rib cross-section from specimen S48 after digital cleaning ............................. 16
Figure 5: Masks used for measurement of total bone area in microns (left) and
tetracycline-labeled bone area in microns (right) for specimen S48 ................................ 17
Figure 6: Frequency of tetracycline-labeled bone area (in microns) by age for both
cemeteries at Kulubnarti (loess line of fit included) ......................................................... 21
x
CHAPTER 1: INTRODUCTION
Purpose
Ancient Nubians were ingesting the antibiotic, tetracycline, likely through beer
(Bassett et al. 1980; Hummert and Van Gerven 1982; Keith and Armelagos 1988;
Armelagos et al. 2001). If tetracycline was ingested through a culturally influenced
activity as part of the daily lives of the ancient Nubians, then the consumption of
tetracycline should vary with cultural-behavioral differences. To test this hypothesis,
amounts of tetracycline labeling of skeletal remains from two contemporaneous
cemeteries were compared. At the site of Kulubnarti, 21-S-46 cemetery (716 CE)
skeletons likely represent a working underclass, and the 21-R-2 cemetery (752 CE)
contains the remains of a land-owning class (Adams and Adams 2007). Interpretations of
archaeological and osteological evidence also suggest that poorer health and higher
mortality occurred in the S population (Van Gerven et al. 1995; Adams and Adams
2007).
Past studies measuring amounts of tetracycline-labeled bone in ancient Nubians,
such as those conducted by and Bassett et al. (1980) and Hummert and Van Gerven
(1982), were limited by available technology and methods of the time. Results of the
current study highlight the merit of reexamination with newer methods; and demonstrate
1
how skeletal biology and bioarchaeology can augment our understanding of biocultural
interactions in the past.
Background of Kulubnarti
The site of Kulubnarti contains two, cotemporaneous, Christian cemeteries dated
to AD 550-800 (Turner et al. 2007) located within the Batn el Hajar (or “belly of rock”)
region of Upper Nubia (Fig. 1). This "barren region of rocks and rapids" extends for 150
miles along the Nile, from the Second Cataract to the Dal Cataract (Van Gerven et al.
1981). Kulubnarti is a penninsula of tall granite outcrops, connected to the west riverbank
of the Nile. In modern times, an island was formed when the peninsula separated from
the mainland by flooding, resulting from construction of the Aswan Dam (Van Gerven et
al. 1981; Campbell Hibbs 2010). Beyond the Nile in this region, the desert is "a virtually
lifeless expanse of rocky jebels (large outcroppings of rock) interspersed with pockets of
sandy wadi" (Van Gerven et al. 1995: 468).
Regional subsistence patterns have changed relatively little over the course of
5,000 years, consisting predominately of small-scale farming villages, which were
marginal at best. Villages depended on retaining walls in order to protect alluvial soils
necessary for agriculture. These soils existed only in pockets and coves, and not as a
continuous floodplain. Crops were irrigated by means of a saqia water wheel (Van
Gerven et al. 1995) and annual flooding (Campbell Hibbs 2010). Staple crops consisted
of beans, barely, lentils, peas, sorghum, millet, dates, and wheat. Domestication of a few
2
animals such as cattle, sheep, and pigs was employed, but animal products appeared to
have been a minor part of the diet (Van Gerven et al. 1995).
Figure 1: Map of Nubia (Turner et al. 2007)
Trace element and isotope analyses, coprolite analyses, and skeletal indicators of
stress suggest the diet at Kulubnarti was reflective of poor agricultural yields (Martin et
3
al. 1989; Sheridan 1992; White et al. 2004; Turner et al. 2007). Meager harvests likely
were due to the inhospitable nature of the Batn el Hajar, low water levels in the Nile, and
general isolation and poverty of the people inhabiting Kulubnarti. This diet has been
estimated to be deficient in vitamins C, B6, B12, folacin, and protein; but high in phytates,
fiber, tannins, and phosphorous, which all inhibit iron uptake (Carlson et al. 1974;
Sheridan 1992; Van Gerven et al. 1995; Turner et al. 2007). Most dietary sources of
protein came from beans and legumes, which are low in iron content in comparison to
animal products. Due to this diet, in conjunction with the high likelihood of parasites,
such as hookworm, which may cause intestinal bleeding, it is probable that the population
suffered from iron-deficiency anemia (Martin et al. 1989; Sheridan 1992; Mittler and Van
Gerven 1994). However, despite widespread poverty and malnutrition of the Kulubnarti
populace, class divisions may still have been present (Van Gerven et al. 1995; Adams
1999; Adams and Adams 2007; Turner et al. 2007).
Ethnographic and historical evidence indicates that within Nubian society are
social distinctions between land-owners and itinerate farmers working the land (Adams
and Adams 2007). Interpretations that the Kulubnarti R and S cemeteries represent
communities of each respective class are supported by archaeological and
paleopathological evidence (Van Gerven et al. 1995; Adams 1999; Adams and Adams
2007; Turner et al. 2007). Archaeological evidence consists primarily of differences in
architecture and burial goods associated with wealth, such as more elaborate textiles
(Adams and Adams 2007; Turner et al. 2007). Higher frequencies of material remains
associated with greater wealth (according to estimated production efforts of particular
4
item qualities) are found at the R cemetery site, and higher frequencies of structures and
lower quality goods, associated with lesser wealth, are found at the S cemetery site
(Adams 1999; Adams and Adams 2007). All skeletal markers of stress indicate that the
remains interred in the S cemetery suffered poorer health than in the R cemetery
community, particularly among sub-adults (Martin et al. 1984; Martin et al. 1989; Mittler
and Van Gerven 1994; Van Gerven et al. 1995). Higher sub-adult mortality rates and
growth retardation, cribra orbitalia lesion frequencies, and distributions of enamel
hypoplasia frequencies have been reported in the S cemetery group compared to the R
(Van Gerven et al. 1981; Martin et al. 1984; Martin et al. 1989; Van Gerven et al. 1990;
Mittler and Van Gerven 1994; Van Gerven et al. 1995).
Health of the Kulubnarti Populace
Cribra orbitalia, a form of porotic hyperostosis associated with childhood
nutritional stress (Martin et al. 1989; Van Gerven et al. 1995), is highly prevalent in
remains from both the R and S cemeteries of Kulubnarti; with more than 80% of
individuals from the Kulubnarti cemeteries exhibiting lesions characteristic of cribra
orbitalia (Mittler and Van Gerven 1994; Van Gerven et al. 1995). This condition is
characterized by porous, pathological lesions in the bones of the orbit which form
through the expansion of bone marrow as the body attempts to compensate for the
inadequate production of red blood cells associated with various forms of anemia,
suffered during childhood stages of growth and development (Lallo et al. 1977; Sheridan
5
1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Wapler et al. 2004; Walker
et al. 2009; Oxenham et al. 2010). Traditionally, the pathology has been associated with
iron-deficiency anemia (Carlson et al. 1974; Lallo et al. 1977; Mittler and Van Gerven
1994; Van Gerven et al. 1995; Oxenham et al. 2010), though arguments have been made
for its attribution to other forms of anemia as well, such as hemolytic or megaloblastic
anemias (Wapler et al. 2004; Walker et al. 2009). These other types of anemia are also
associated with nutritional deficiencies and are able to produce the sort of marrow
hypertrophy characteristic of porotic hyperostosis (Walker et al. 2009). However,
regardless of the type of anemia, the presence of cribra orbitalia is associated with
childhood anemias and malnutrition; from which the Kulubnarti populations likely
suffered (Sheridan 1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Turner
et al. 2007).
Further indicators of dietary stress found at Kulubnarti include enamel
hypoplasias and osteoporosis. Enamel hypoplasias represent episodes of childhood
nutritional stress resulting in the disruption of ameloblast activity causing zones of
enamel to be reduced in thickness (Van Gerven et al. 1995). Every individual at
Kulubnarti exhibited evidence of at least one hypoplasmic event. In addition, the dental
age in 70.5% of individuals at Kulubnarti exceeded their estimated age as reflected by
other skeletal markers, suggesting that there had been significant skeletal growth
retardation during development (Van Gerven et al. 1995). It has also been suggested that
the Kulubnarti diet may have been deficient in calcium (Sheridan 1992). While calcium
deficiency may have impacted health aspects such as bone tissue production and
6
reproductive capabilities, it would not affect percentages of bone labeled with
tetracycline.
However, several cultural factors may have actually served as buffers to the
morbidity of the Kulubnarti populations. For instance, Campbell Hibbs (2010) suggests
that irrigation practices may have buffered infection rates of schistosomiasis at
Kulubnarti. During the Christian period, it is likely that most irrigation at Kulubnarti was
a result of annual flooding, rather than irrigation canals. While saqia water wheels were
used by the end of the Christian period, there is little to no evidence of their usage before
this time. Lack of irrigation canals and standing water therefore reduced the chance of
exposure to water-born parasites - especially schistosomiasis (a deadly parasite carried by
host snails) - at Kulubnarti, compared to other ancient Nubian sites. Campbell Hibbs
(2010) detected significantly fewer antigens for Schistosoma mansoni in desiccated tissue
samples from Kulubnarti than in Wadi Halfa samples. Infection from schistosomiasis
would have further compounded the effects of malnutrition causing additional diarrhea,
growth retardation, more severe symptoms of anemia, and a variety of other disabling
factors (Campbell Hibbs 2010).
Nubian Antibiotics
Evidence of consumption of the broad spectrum antibiotic, tetracycline, has been
observed in Nubian remains from the sites of Kulubnarti and Wadi Halfa (Bassett et al.
1980; Hummert and Van Gerven 1982; Armelagos et al. 2001). "Rediscovered" in the
7
modern era, tetracycline molecules (Fig. 2) are composed of four six-membered rings
fused linearly - to which a variety of functional groups attach – with characteristic double
bond arrangements (Frost et al. 1961; Dürckheimer 1975; Chopra and Roberts 2001)
Tetracycline binds to calcium when present and therefore labels bone undergoing
mineralization at the time of ingestion. This labeling appears as a yellow-green
fluorescence when thin sections of bone are viewed microscopically under an ultraviolet
light at 490 nanometers (Bassett et al. 1980; Armelagos et al. 2001). Since fluorescence
patterns indicate that osteons were labeled at various times during their formation, and
the remains do not show any signs of post-mortem mold infestation, it is likely that
tetracycline was ingested in vivo (Keith and Armelagos 1988).
N(CH3)2
H
OH
CH3
H
H
OH
CONH2
OH
O
OH
OH
O
Figure 2: Tetracycline molecule
8
The bacterium which produces tetracycline, Streptomycetes, flourishes in warm,
dry, and alkaline environments. Therefore, mud pots in which ancient Nubians stored
grain for brewing beer, produced a perfect environment for cultivating Streptomycetes. In
addition, Streptomycetes comprises 60 to 70% of soil bacteria in Sudanese Nubia.
Consumption of beer seems the ideal conduit for tetracycline ingestion and is favored
over an alternative hypothesis of eating bread baked with Streptomycetes contaminated
grain during a food shortage. This suggestion is based on the nature of tetracycline with
regard to its production and how it labels bone. Streptomycetes will only secrete
tetracycline under harsh environmental conditions, such as those created by fermentation
processes (Armelagos et al. 2001). Generally, 250 mg of tetracycline is considered a safe
therapeutic dosage in modern medicine (Frost et al. 1961; Delaney et al. 1974; Sauer
1976; Basset et al. 1980; Martin et al. 1989); and only 1 to 2 grams of tetracycline per
day are required to produce fluorescence in human bones (Armelagos et al. 2001; Fabsits
2008). Any observable labeling in human bone must therefore result from ingesting
tetracycline at therapeutic dosages. Thus, the amount of bone labeled by tetracycline at
Kulubnarti appears to be a level of intake too high to have occurred from chance
exposure, but rather appears to arise from a culturally sanctioned activity (Bassett et al.
1980).
9
Why Study Tetracycline Consumption at Kulubnarti?
For years bioarchaeologists and paleopathologists in particular, have studied
health and its cultural implications in ancient populations from patterns of skeletal
morphologies indicative of nutritional status, activity levels, morbidity and mortality
(Larsen 1987; Goodman 1993; Armelagos 2003; Armelagos and Van Gerven 2003).
However, information which can be obtained through analyses of skeletal indicators of
health is often complicated and limited. These skeletal markers are often only present in
individuals who survived a disease or period of nutritional stress long enough for the
stressor to leave its mark in bone (Goodman 1993; Ortner 2003). Bioarchaeologists,
therefore, must rely on the general patterns of health indicators which emerge from the
skeletal analysis of a variety of morphological and histological features observed within
an archaeological population in order to draw conclusions (Goodman 1993; Armelagos
2003; Armelagos and Van Gerven 2003).
The presence of tetracycline labeled bone provides a unique opportunity for
studying health in ancient Nubian populations. Since tetracycline and its effects on the
body are able to be studied in modern clinical settings, any influence tetracycline has on
health in modern populations can be extrapolated to ancient populations, even when
physical evidence is limited. Such analogies are possible provided the environments in
which effects are produced are similar. However, when used with other skeletal
indicators relating to health and biocultural interactions, the environmental context in
which the population lived can be reconstructed (Goodman 1993; Armelagos 2003;
Armelagos and Van Gerven 2003; Larsen 2015). Understanding past biocultural
10
interactions in particular ecological contexts can then be applied to understand biocultural
interactions in living populations. Results from this project may also be used in future
research to further understand how tetracycline affects human health in nutritionally
challenging environments.
Summary
The ancient Nubians of Kulubnarti were a marginal farming community of the
inhospitable Batn el Hajar region. Freehold farmers and itinerate sharecroppers were
buried with their peers in two separate, Christian cemeteries in association with material
remains reflective of their status. Despite the generally poor diet of this peasant
community, there is skeletal evidence indicating that the sharecroppers were even more
nutritionally stressed than their land-owning neighbors. Additionally, both cohorts exhibit
evidence of having ingested the antibiotic tetracycline – likely through their dietary
staple, beer. Whether or not social status influenced access to tetracycline is under
investigation in this study.
11
CHAPTER 2: MATERIALS AND METHODS
Materials
Thirty-eight thin rib cross-sections from Kulubnarti were analyzed. Previously,
rib samples were embedded in epoxy resin and ground down to a thickness
approximating 100 micrometers. S and R cemetery sub-group samples consisted of 19
single burials (n = 38), matched for sex and age as closely as possible using available
slides and data (Table 1). Aided by the remarkable preservation and often natural
mummification of the remains, age at death and sex were estimated utilizing several
methods, detailed by Van Gerven et al. (1981). These methods included visual
examination of preserved external genitalia, pelvic morphologies, epiphyseal unions,
dental eruptions, and changes in the os pubis. Eleven sub-adults age 16 or younger and
eight adults were selected from each cemetery. Sub-adult females were under-represented
in the sample. This under-representation likely stems from difficulties estimating the sex
for pre-pubescent skeletal remains as noted by Phenice (1967) and varying degrees of
natural mummification. Comparative analysis of the sub-adult populations by sex is
therefore not presented. Sample size was limited to n=38 by access to materials and
funding.
12
Table 1: Sample specimens and associated sex and age estimations from Kulubnarti
Specimens
by Burial
Number
Sex
Age
Specimens
by Burial
Number
Sex
Age
S13
male
12 years
R68
male
12 years
S62
male
14 years
R146
male
15 years
S68 a
male
4 years
R147
male
16 years
S236
male
16 years
R196
male
1.5 years
S36
?
13 years
R18
female
15 years
S23
?
5 years
R84
female
2 years
S28
?
7 years
R192
female
16 years
S37
?
1 year
R21
?
6 years
S41
?
15 years
R142
?
9 years
S47
?
8 years
R179
?
14 years
S48
?
11 years
R201
?
7 years
S16
male
27 years
R15
male
22 years
S99
male
39 years
R34
male
31 years
S173
male
42 years
R158
male
19 years
S206
male
37 years
R163
male
21 years
S1
female
42 years
R72
female
38 years
S109
female
31 years
R107
female
51+ years
S186
female
31 years
R122
female
27 years
S207
female
49 years
R144
female
34 years
13
Methods
Thin sections were viewed under an ultraviolet light microscope at 490 nm. At
this intensity, fluorophors in bone labeled with tetracycline fluoresce yellow-green (Frost
1969; Bassett et al. 1980; Armelagos et al. 2001). Amount of tetracycline-labeled bone
was quantified by area and labeled osteon frequencies using three measurement methods.
Labeled-bone area was determined through application of a new image analysis
technique and point-count methods. Then labeled osteon frequencies were determined by
manually counting fluorescing osteons – similar to the osteon count methods employed in
prior studies of tetracycline labeling in Nubian populations (Bassett et al. 1980; Hummert
and Van Gerven 1982; Fabsits 2008). However, this technique does not count lamellar or
trabecular tetracycline-labeled bone tissues or partially resorbed labeled osteons.
Therefore, a new technique of image analysis - described below - was developed to
directly measure labeled bone area. Additionally, tetracycline-labeled bone area was
estimated using a point-count method, commonly used to study bone histology (Frost
1969).
New Technique of Image Analysis Method
A video motion camera was mounted to a UV light microscope with a 0.63x lens
attached. Photographs were streamed from the microscope camera to a computer using
Image-Pro Plus 7.0. Then a series of images spanning entire rib cross-sections under the
5x optical lens were photographed. Image brightness and contrast were set to 33, default
enhancements; maintaining section photograph qualities were as similar to their actual
14
appearance (observed directly through the microscope) as possible and contained
minimal background noise.
Photographs were imported into Microsoft Image Composite Editor (ICE) and
each series of images stitched together to produce a single composite image of each rib
cross-section (Fig. 3). Adobe Photoshop was used to “digitally clean” these images of
soft tissue attached to the bone and background colorations (as much as possible) to
minimize interference during image analysis (Fig. 4). Cross-section photographs were
archived before and after digital cleaning.
Figure 3: Rib cross-section from specimen S48 before digital cleaning
15
Figure 4: Rib cross-section from specimen S48 after digital cleaning
To distinguish tetracycline-labeled bone tissue, two masks of the cross-section
were created for each slide. One mask selected all pixels of bone tissue, the second only
pixels of bone tissue labeled with tetracycline (Fig. 5). Tissue differentiation was
accomplished using color bitmap manipulation in which ranges of green intensity levels
were selected for mask production. Red and blue intensity levels were set to zero. Since
each bone cross-section and image differed in preservation quality and UV light exposure
time - which affected the intensity of yellow-green tetracycline fluorescence (Keith and
Armelagos 1988; Maggiano et al. 2006) - unique green color ranges had to be manually
set to select the correct tissue areas for each mask. The total mask area in micrometers
was then measured using Image-Pro. Total bone and total tetracycline-labeled bone areas
for each cross-section were obtained, and percentages of tetracycline-labeled bone were
calculated.
16
Figure 5: Masks used for measurement of total bone area in microns (left) and
tetracycline-labeled bone area in microns (right) for specimen S48
Osteon Count Method
Digitally cleaned images were then reopened in Adobe Photoshop, and its
counting tool was used to track manual osteon selections. All osteons containing a
Haversian canal were counted. Osteons visually determined to be over 50% labeled were
then counted separately. Most tetracycline-labeled osteons were not homogenously
labeled. However, this pattern is expected if tetracycline consumption was not restricted
to the average osteon formation period of 80 days. Therefore, it was not considered
necessary to count only fully labeled osteons. Osteon counts were recorded and labeledosteon percentages calculated.
Point-Count Method
Image-Pro was used to create a grid overlay containing at least 100 intersections
sized to each individual cross-section image. The Image-Pro counting tool was then used
17
to track all grid intersections over bone and then over tetracycline-labeled bone tissue.
Resulting counts, grid sizes, and percent intersections over labeled bone calculations for
individual cross-sections were also recorded in Excel.
Descriptive statistics of results for key variables for each method of measurement
were determined (Table 2). Visual examinations suggested that variables are not
distributed normally and therefore require the use of non-parametric statistical tests. The
statistical significance of differences in tetracycline-labeled bone content between the
cemeteries were evaluated using the non-parametric Mann-Whitney test and a 95%
confidence level (p ≤ 0.05).
Summary
For 38 individuals from both Kulubnarti cemeteries, three measurements were
taken under UV light from thin rib cross-sections to quantify amounts of bone labeled
with tetracycline. A new technique was developed to measure labeling directly by area
and two established techniques - counting osteons and labeled points - were also
employed for comparison. Mann-Whitney U tests were used to compare amounts of
tetracycline-labeled bone between the cemeteries at the 95% confidence interval (p ≤
0.05) for each method of measurement.
18
CHAPTER 3: RESULTS
In all cases, regardless of whether tetracycline-labeled bone was quantified via
area or osteon frequencies, no significant differences in percentages of tetracyclinelabeled bone tissue were observed between S and R cemeteries (U: p ≤ 0.05) (Table 2). In
addition, results obtained using the new technique of image analysis to measure total area
of tetracycline-labeled bone were similar those obtained using previously established
methods. However, when percent area of labeled bone tissue was graphed against
estimated age at death, a different pattern for percent labeled bone tissue between the two
cemeteries - especially for sub-adults - was revealed (Fig. 6). The amount of labeled bone
tissue for sub-adults increased with age in the R cemetery, while it decreased in
individuals from the S cemetery. After the age of approximately 15 years, the ageassociated patterns for tetracycline labeling for both cemetery samples begin to follow a
similar pattern of relatively gradual increase with age.
19
Table 2: Percent averages of tetracycline-labeled bone for all ages at Kulubnarti
Cemetery
% Labeled
Bone Area
S
19
21.595
26.494 14.930
% Labeled
Bone Area
R
19
27.669
26.599 19.044 178.00 - 0.073
% Labeled
Bone Area
Combined
38
23.660
26.547 16.878
% Labeled
Osteons
S
19
21.693
26.730 14.549
% Labeled
Osteons
R
19
27.826
25.103 15.445 162.00 - 0.540
% Labeled
Osteons
Combined
38
25.741
25.917 14.822
S
19
24.490
25.848 14.095
R
19
23.423
24.046 16.169 166.00
Combined
38
23.957
24.947 14.989
% Labeled
Grid
Intersections
% Labeled
Grid
Intersections
% Labeled
Grid
Intersections
*
n
Median
Mean
Std.
Dev.
Measurement
U
z
-0.423
p*
0.954
0.603
0.686
Mann-Whitney test statistic for cemetery comparison, analyzed using a 95% confidence
interval
20
Figure 6: Frequency of tetracycline-labeled bone area (in microns) by age for both
cemeteries at Kulubnarti (loess line of fit included)
Observed differences in the patterns for tetracycline labeling between the cemeteries for
sub-adults may reveal differences in diet, especially breast feeding behavior. Tetracycline
can be passed from mother to child through breast-feeding (Chopra and Roberts 2001;
Sánchez et al. 2004). To control for possible effects of mother-offspring transmission of
tetracycline on the total amount of labeled bone, and account for differences in
tetracycline-labeled bone vary between cemeteries, Mann-Whitney tests were applied to
21
samples of only individuals age 12 years and older. By age 12, bone turnover should
completely replace any bone formed during breast-feeding (Stout p.c. 2014). Again, no
significant differences (U: p ≤ 0.05) in tetracycline-labeled bone content between
cemeteries were observed (Table 3). Small sample size precluded testing only individuals
estimated to be younger than age 12 at death for differences in tetracycline-labeled bone
between the cemeteries.
Summary
Regardless of which measurement technique was used, there are no significant
differences in the amounts of tetracycline-labeled bone between the cemeteries at the
95% confidence level. Whether there is a difference between sub-adults and adults within
cemeteries, or sub-adults between the cemeteries, requires an expansion of the sample to
test. However, it does not appear that there is a significant difference in tetracycline
consumption between the adults of each cemetery.
22
Table 3: Percent averages of tetracycline-labeled bone among those aged 12 and older at
Kulubnarti
Measurement
Cemetery
n
Median
Mean
Std.
Dev.
% Labeled
Bone Area
S
13
18.016
22.225
14.891
% Labeled
Bone Area
R
14
29.182
29.126
18.749
% Labeled
Bone Area
Combined
27
20.248
25.803
17.043
% Labeled
Osteons
S
13
17.526
21.985
13.132
% Labeled
Osteons
R
14
27.078
26.512
14.949
% Labeled
Osteons
Combined
27
21.004
24.332
14.022
% Labeled
Grid
Intersections
S
13
18.452
21.494
12.961
% Labeled
Grid
Intersections
R
14
25.448
25.779
16.054
% Labeled
Grid
Intersections
Combined
27
21.324
23.716
14.531
*
U
z
p*
72.00 - 0.922
0.375
81.00 - 0.485
0.650
79.00 - 0.582
0.583
Mann-Whitney test statistic for cemetery comparison, analyzed using a 95% confidence
interval
23
CHAPTER 4: DISCUSSION
The use of image analysis technology to measure tetracycline labeling in terms of
labeled bone area proved to be appropriate, and comparable to other methods. Observed
small differences of 1-2 percentage points between the three measurement techniques
(Table 2) likely result from what is actually being measured by each method. Where the
proposed image analysis technique and point count methods measures area of
tetracycline-labeled bone, the osteon count method only provides a frequency of labeled
osteons; and does not include other types of bone tissue, such as primary lamellar bone,
or partially resorbed secondary bone of remodeled fragmentary osteons. Results of this
study also suggest that the point count method can serve as a substitute for area
measurements, when technology does not make direct tetracycline-labeled bone area
measurements feasible.
Means for tetracycline-labeled bone for each method of measurement fall within
the range of 24-27% of bone tissue being labeled (Table 2). The amount of bone labeled
with tetracycline in the Kulubnarti Nubian populations supports the assumption that
tetracycline ingestion by means of a common, characteristic activity, exceeding a
therapeutic threshold. Beer consumption (a culturally influenced activity) continues to be
the most probable source of tetracycline for ancient Nubian populations (Bassett et al.
24
1980; Armelagos et al. 2001). Because beer was a staple of the Nubian diet (Bassett et al.
1980), it follows that all members of society, regardless of class, would have access to
beer. Unless beer was used in a ritual context beyond nutritional sustenance, one would
expect access to beer to be mostly uniform across classes. Thus, if tetracycline was
consumed through beer, the amount of labeled bone should be homogeneous across
groups, which is the case at Kulubnarti. Despite the S cemetery exhibiting poorer health
and less material wealth than the R cemetery (according to archaeological and
paleopathological findings (Sheridan 1992; Mittler and Van Gerven 1994; Van Gerven et
al. 1995; Adams and Adams 2007; Turner et al. 2007)), there are no significant
differences in tetracycline consumption. Therefore, the classed communities (represented
by the S and R cemeteries at Kulubnarti) likely had equal access to beer. Future sample
expansions are needed to further investigate age trends in tetracycline consumption.
Other hypotheses have been posed regarding how tetracycline was ingested or
whether the fluorescence observed in Nubian remains is from tetracycline in the first
place. Pipenbrink (1983, 1986) suggests that mold infestation during diagenesis could be
responsible for the yellow-green fluorescence observed in Nubian samples. However,
characteristic tunneling, cuffing, and fluorescence in wake of this microbial destruction
(Pipenbrink 1986) are not observed in the Kulubnarti sample; nor in the ancient Nubian
NAX population from Wadi Halfa (Bassett et al. 1980; Keith and Armelagos 1988;
Nelson et al. 2010). Observed osteon fluorescence appears as more discrete patterns
rather than diffused (a common pattern of fluorescence in skeletal remains when caused
by mold infestation (Pipenbrink 1986; Keith and Armelagos 1988)). In addition, labeling
25
does not only appear as surface labeling or staining, but embedded internal
microstructures of bone are labeled as well. Surface-only labeling is reflective of in vitro
labeling (Pipenbrink 1986; Keith and Armelagos 1988) and this pattern is not present in
the Kulubnarti sample. Pipenbrink (1983, 1986) was not convinced that published
photographs of fluorescing osteons from Nubian sites were not actually surface labels.
This skepticism may be due in part to technology available at the time, preventing
photography and publication of the entire bone cross-section (Bassett et al. 1980; Keith
and Armelagos 1988). By incorporating new technology in developing updated methods,
it is clear that bone material is labeled throughout the bone, rather than just the surface
areas (Figs. 1-2). However, successful extraction of tetracycline from the NAX
population is the most conclusive evidence that fluorescence in ancient Nubian skeletal
remains is from tetracycline consumed in vivo (Nelson et al. 2010).
Tetracycline was also not likely consumed by eating mold-infested or
Streptomycetes contaminated grains (made into foodstuffs such as bread) during a food
shortage. First of all, tetracycline is only secreted under harsh conditions, such as
fermentation (Armelagos et al. 2001; Maggiano et al. 2003; Nelson et al. 2010). Thus,
foodstuffs would not have regularly produced the necessary environment for secretion of
this antibiotic. Secondly, even if tetracycline secretion had been managed, eating
contaminated grains during a food shortage would have produced sporadic labeling.
Observed patterns of tetracycline-labeled bone fluorescence indicate that the drug was
ingested on a regular basis (Bassett et al. 1980; Keith and Armelagos 1988; Armelagos et
al. 2001; Nelson et al. 2010). High percentages of tetracycline-labeled bone content
26
resulting from this project further support this conclusion suggesting that tetracycline was
ingested often. Maggiano et al. (2003) also reached the conclusion that tetracycline was
ingested in vivo near the Dakhleh Oasis, although the authors argued that contaminated
foodstuffs were the source of tetracycline. Their main argument was that contaminated
and spoiling grains were the source of tetracycline in contrast to ale. This conclusion
centers mainly on their assumption that without controlling the amount of Streptomycetes
present during fermentation, the resulting beverage would be “foul-tasting” (Maggiano et
al. 2003: 341). Taste is however, a subjective quality that may be influenced by cultural
preferences. Without having an exact recipe used for ancient Nubian beer brewing or
knowledge of cultural notions regarding food, such an argument does not adequately
counter the apparent regularity of tetracycline consumption; which is greater than would
be expected if spoiling food had been the source.
Tetracycline-labeled bone percentages resulting from this study (Table 2) are
similar to previous findings of Bassett et al. (1980), where about 30% of all observed
osteons from the ancient Sudanese-Nubian X-Group population exhibited some level of
fluorescence from tetracycline labeling. However, the results differ about eightfold from
those reported by Hummert and Van Gerven (1982) for the Kulubnarti population (3.6%
of osteons were labeled with tetracycline). These discrepancies are likely explained by
the prior study using thin bone sections from metacarpals and femurs rather than ribs and
differences in slide preparation techniques. Their slides were prepared by a different
technician using currently available, but different technologies and measurement
methods. The technology available in 1982 also limited the scope of measurements,
27
making sub-sampling cross-sections the only practical means of measurement. By subsampling larger bones, the portions of cross-sections measured for labeled osteon
frequencies may have simply had fewer osteons than elsewhere on the same cross-section
and/or what would be expected in a comparable sub-sample from ribs. Interestingly,
Hummert and Van Gerven (1982) found that only 63% of their sample contained
fluorophors (41 of the 110 samples contained no tetracycline labeling) as opposed to the
100% reported by Bassett et al. (1980) for the X-Group. In preparation for conducting
this study on the Kulubnarti populations, of 182 individuals from which rib slides were
created (and from which samples were used for this study), only six had little to no
tetracycline labeling. However, despite discrepancies in resulting means of tetracyclinelabeled bone between studies, Hummert and Van Gerven (1982) also found there was not
a significant difference in amount of labeled bone between the S and R cemeteries.
The results also differ from those that were previously reported (Margolis et al.
2013) for this sample. After reexamination of data and materials, it became apparent that
the standard UV light alignment settings had slipped during cross-section photography.
Which cross-section photos were affected by this setting change were determined based
on visual appearance of photographs in conjunction with the timing of the original
settings slip and order in which photographs were stitched together (as were recorded in
laboratory notes). All cross-section photographs affected by the change of settings were
reproduced using proper UV light alignment settings and data re-analyzed. In addition,
the hit-and-miss method and more appropriate, non-parametric statistical testing have
been employed in this updated analysis.
28
Since the sample size was relatively small, a future expansion of the sample may
provide a more accurate estimate of tetracycline consumption. To further investigate why
there are opposing trends in accumulation of tetracycline labeling between the cemetery
communities, sample size expansion would be particularly important. Sample expansion
would help determine if the observed trend opposition resulted from small sample size or
is real. Additional research on tetracycline-labeled bone accumulation trends in the
Kulubnarti cemeteries will also aid in examination of whether beer (as the conduit of
tetracycline consumption) was a dietary staple or was used in different contexts according
to class. Data obtained from this thesis however, may be used to better understand health
in the Kulubnarti, Nubian population and why certain skeletal pathology trends, such as
infection rates, are observed.
Summary
Resulting means of tetracycline-labeled bone in the Kulubnarti cemeteries are
similar to those reported for the X-Group at Wadi Halfa, but differ greatly from those
previously reported for Kulubnarti (Bassett et al. 1980; Hummert and Van Gerven 1982).
These discrepancies are likely explained by differences in measurement methods as
constrained by available technology. However, as was previously concluded by Hummert
and Van Gerven (1982), there are no significant differences in tetracycline consumption
between the cemeteries at Kulubnarti, despite marked differences in skeletal indicators of
stress and status. Due to labeling patterns, the high percentages of labeled-bone, and
29
nature of tetracycline production, it is likely that the ancient Nubians were ingesting
tetracycline through culturally characteristic beer drinking.
30
CHAPTER 5: CONCLUSIONS
High percentages of bone labeled with tetracycline in the Kulubnarti Nubian
populations offers more support to the hypothesis that tetracycline was ingested in
therapeutic doses in vivo by means of a common, culturally characteristic activity. The
hypothesized means of ingestion for the antibiotic, as a culturally influenced activity,
through consumption of beer (Bassett et al. 1980; Armelagos et al. 2001), continues to be
the most probable source of tetracycline for the ancient Nubian populations. However,
the results from this study are surprising as the skeletal indicators of health and
archaeological evidence between the two cemeteries are markedly different (Sheridan
1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Adams and Adams 2007;
Turner et al. 2007). Despite the S cemetery exhibiting poorer health and less material
wealth than the R cemetery, the two groups appear to have been equal in regards to their
tetracycline consumption. While it is still likely that the S cemetery represents an
underclass to those interred in the R cemetery (Adams and Adams 2007), it can be
concluded that class differences were not mediating access to tetracycline, and in turn,
beer. In addition, the new method of measuring tetracycline-labeled bone area using
image analysis (as presented in this thesis) provides an efficient and comprehensive
procedure for determining tetracycline-labeled bone content. This methodology will be
31
beneficial not only for bioarchaeological studies on health in ancient populations, but for
any skeletal histological research incorporating use of tetracycline as a marker of bone
growth and resorption, and clinical studies on the physiological effects of this antibiotic.
32
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36
APPENDIX A: RAW DATA
Table 4: Kulubnarti cemetery 21-S-46 (S Cemetery) raw data
Green
Total
Green
Total
color range Area of
color range Area of
(Total
Labeled
(Total bone Bone (in
labeled
Bone (in
area)
microns)
bone)
microns)
Specimens
by Burial #
Sex
Age
S13
male
12 years
62-255
% Bone
Labeled
# of
Osteons
# of
Mostly
Labeled
Osteons
Grid Used
(in
microns)
% Osteons Total Hits Labeled
Labeled and Misses
Hits
% Labeled
Hits
39
37.86408 400x400
5.940594 500x500
15140824
112-255 5334595.5 35.233191
208
92
44.23077
103
37
S62
male
14 years
33-255
23732366
76-255
941806.31 3.9684468
183
11
6.010929
101
6
S68 a
male
4 years
47-255
6590091.5
77-255
1423142.6 21.595187
46
9
19.56522
100
21
S236
male
16 years
48-255
29471674
81-255
18.015468
189
41
21.69312
117
23
19.65812 500x500
S36
?
13 years
50-255
17232128
82-255
2823355.8 16.384255
73
22
30.13699
111
15
13.51351 400x400
S23
?
5 years
65-255
10136109
10-255
44.04337
99
47
47.47475
112
48
42.85714 300x300
S28
?
7 years
55-255
15105379
98-255
4027931.8 26.665546
81
23
28.39506
98
24
24.4898
S37
?
1 year
51-255
6146576.5
92-255
2662261.3 43.312912
71
32
45.07042
107
50
46.72897 250x250
S41
?
15 years
45-255
24893706
80-255
2721774.3 10.933584
194
34
17.52577
133
20
15.03759 450x450
S47
?
8 years
86-255
18928696
147-255
5744530
30.348261
190
57
30
96
26
27.08333 450x450
S48
?
11 years
96-255
23435542
158-255
11367031 48.503384
192
99
51.5625
109
54
49.54128 475x475
S16
male
27 years
60-255
19538928
98-255
3283693.8 16.805906
263
36
13.68821
115
16
13.91304 425x425
S99
male
39 years
74-255
30002128
128-255 2405812.5 8.0188062
304
31
10.19737
129
6
4.651163 500x500
S173
male
42 years
67-255
28764506
103-255 7399316.5 25.723774
314
52
16.56051
129
33
25.5814
S206
male
37 years
73-255
13945925
129-255 2879463.5 20.647347
87
14
16.09195
166
43
25.90361 300x300
S1
female
42 years
83-255
31431152
150-255
17668838 56.214414
290
105
36.2069
129
59
45.73643 500x500
S109
female
31 years
68-255
18781614
114-255 8293123.5 44.155542
157
71
45.22293
97
39
40.20619 450x450
S186
female
31 years
65-255
19293838
102-255 2425776.8 12.572806
235
17
7.234043
108
14
12.96296 450x450
S207
female
49 years
95-255
18376276
151-255 3720810.8 20.247904
219
46
21.00457
168
31
18.45238 350x350
5309460
4464284
37
21
275x275
425x425
500x500
APPENDIX A: RAW DATA
Table 5: Kulubnarti cemetery 21-R-2 (R Cemetery) raw data
Green
Total
Green
Total
color range Area of
color range Area of
(Total
Labeled
(Total bone Bone (in
labeled
Bone (in
area)
microns)
bone)
microns)
38
% Bone
Labeled
# of
Osteons
# of
Mostly
Labeled
Osteons
36.516239
165
160-255
23443904 61.795048
165-255
19131964 49.921439
11313690
26986584
60-255
16 years
6 years
?
R179
R201
Grid Used
(in
microns)
% Osteons Total Hits Labeled
Labeled and Misses
Hits
% Labeled
Hits
72
43.63636
111
26
23.42342 450x450
207
101
48.79227
196
108
55.10204 450x450
367
159
43.32425
208
79
37.98077 450x450
140-255 149101.77 1.3178881
315
13
4.126984
104
4
3.846154 350x350
153-255
11987440 44.419998
267
96
35.95506
229
91
39.73799 350x350
12388930
139-255 217328.91 1.7542186
149
0
0
102
3
2.941176 350x350
76-255
36035896
128-255
19281566 53.506554
361
159
44.04432
152
73
48.02632 500x500
69-255
14038496
140-255 2150403.5 15.317905
115
32
27.82609
119
22
18.48739 350x350
9 years
87-255
20773254
155-255
10007632 48.175563
186
66
35.48387
103
46
44.66019 450x450
?
14 years
72-255
31230090
141-255 2111030.8 6.7596052
219
24
10.9589
133
7
5.263158 500x500
?
7 years
60-255
10227909
105-255 3176060.8 31.052885
73
28
38.35616
96
25
26.04167 350x350
R15
male
22 years
46-255
35485164
79-255
16.509175
322
49
15.21739
142
14
9.859155 500x500
R34
male
31 years
28-255
25347862
70-255
1045681.8 4.1253254
236
23
9.745763
137
9
6.569343 450x450
R158
male
19 years
28-255
21047066
54-255
2885944
13.711859
125
7
5.6
106
10
9.433962 450x450
R163
male
21 years
91-255
32027640
148-255
6435213
20.092686
186
44
23.65591
136
29
21.32353 500x500
R72
female
38 years
82-255
18889880
129-255
6794993
35.971605
200
61
30.5
100
36
R107
female 51+ years
58-255
25045434
115-255 6929769.5 27.668794
395
54
13.67089
131
38
29.00763 450x450
R122
female
27 years
73-255
15637168
141-255 4799962.5 30.695856
160
50
31.25
91
25
27.47253 450x450
R144
female
34 years
26-255
16346545
66-255
162
24
14.81481
94
11
11.70213 450x450
Specimens
by Burial #
Sex
Age
R68
male
12 years
86-255
20606906
123-255
7524867
R146
male
15 years
89-255
37938160
R147
male
16 years
96-255
38324144
R196
male
1.5 years
66-255
R18
female
15 years
93-255
R84
female
2 years
R192
female
R21
?
R142
5858308
991312.5 6.0643549
38
36
450x450
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
S1
S13
39
S16
S23
S28
39
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
S36
S37
40
S41
S47
S48
40
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
S62
S68a
41
S99
S109
S173
41
Total Bone Mask
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
S186
S206
42
S207
S236
42
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
R15
R18
43
R21
R34
R68
43
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
R72
R84
44
R107
R122
R142
44
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
R144
R146
45
R147
R158
R163
45
Labeled Bone Mask
APPENDIX B: RIB CROSS-SECTION PHOTOS
Specimen
Burial #
Stitched Cross-Section
Cross-Section After
Digital Clean-Up
Total Bone Mask
R179
46
R192
R196
R201
46
Labeled Bone Mask
47